SG188121A1 - Compositions and methods for inhibiting expression of eg5 and vegf genes - Google Patents

Abstract

This invention relates to compositions containing double-stranded ribonucleic acid(dsRNA) in a SNALP formulation, and methods of using the compositions to inhibit the expression of the Eg5 and Vascular Endothelial Growth Factor (VEGF), and methods of using the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.(No suitable figure)

Description

COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF Eos

AND VEGF GENES

} Related Applications

This application claims the benefit of U.S. Provisional Application No. 61/034,019, filed March 5, 2008, and U.S. Provisional Application No. 61/083,367, filed July 24, 2008, and U.S. Provisional Application No. 61/086,381, filed August 5, 2008, and U.S. Provisional

Application No. 61/112,079, filed November 6, 2008, and U.S. Provisional Application No. 61/150,664, filed February 6, 2009, which are hereby incorporated in their entirety by reference. :

Field of the Invention

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA), and their use in mediating RNA interference to inhibit the expression of a combination of genes, e.g., the EgS and Vascular Endothelial Growth Factor (VEGF) genes formulated in SNALP, and the use of the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.

Background of the Invention

The maintenance of cell populations within an organism is governed by the cellular processes of cell division and programmed cell death. Within normal cells, the cellular events associated with the initiation and completion of each process is highty regulated. In proliferative disease such as cancer, one or both of these processes may be perturbed. For example, a cancer cell may have lost its regulation (checkpoint control) of the cell division cycle through either the overexpression of a positive regulator or the loss of a negative regulator, perhaps by mutation.

Alternatively, a cancer cell may have lost the ability to undergo programmed cell death through the overexpression of a negative regulator. Hence, there is a need to develop new chemotherapeutic drugs that will restore the processes of checkpoint control and programmed cell death to cancerous cells.

One approach to the treatment of human cancers is to target a protein that is essential for cell cycle progression, In order for the celi cycle to proceed from one phase to the next, certain prerequisite events must be completed. There are checkpoints within the cell cycle that enforce the proper order of events and phases. One such checkpoint is the spindie checkpoint that occurs during the metaphase stage of mitosis. Small melecules that target proteins with essential functions in mitosis may initiate the spindle checkpoint to arrest cells in mitosis. Of the small molecules that arrest cells in mitosis, those which display anti-tumor activity in the clinic also induce apoptosis, the morphological changes associated with programmed cell death. An effective chemotherapeutic for the treatment of cancer may thus be one which induces checkpoint control and programmed cell death. Unfortunately, there are few compounds available for controlling these processes within the cell. Most compounds known to cause mitotic arrest and apoptosis act as tubulin binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function/structure of the mitotic spindle thereby causing mitotic arrest. Because most of these compounds specifically target the tubulin protein which is a component of all microtubules, they may also affect one or more of the numerous normal cellular processes in which microtubules have a role. Hence, there is also a need for agents that more specifically target proteins associated with proliferating cells.

Eg5 is one of several kinesin-like motor proteins that are localized to the mitotic spindle and known to be required for formation and/or function of the bipolar mitotic spindle.

Recently, there was a report of a small molecule that disturbs bipolarity of the mitotic spindle (Mayer, T. U. et, al. 1999. Science 286(5441) 671-4, herein incorporated by reference). More specifically, the small molecule induced the formation of an aberrant mitotic spindle wherein a monoastral array of microtubules emanated from a central pair of centrosomes, with chromosomes attached to the distal ends of the microtubules, The small molecule was dubbed "monastrol” after the monoastral array. This monoastral array phenotype had been previously observed in mitotic cells that were immunodepleted of the Eg5 motor protein. This distinctive monoastral array phenotype facilitated identification of monastrol as a potential inhibitor of

Eg5. Indeed, monastrol was further shown to inhibit the Fg§ motor-driven motility of microtubules in an in vitro assay. The Eg inhibitor monastrol had no apparent effect upon the related kinesin motor or upon the motor(s) responsible for golgi apparatus movement within the cell. Cells that display the monoastral array phenotype either through immunodepletion of Eg5 or monastrol inhibition of EgJ arrest in M-phase of the cell cycle.

However, the mitotic arrest induced by either immunodepletion or inhibition of EgS is trangient (Kapoor, T. M., 2000. J Cell Biol 150(5) 975-80). Both the monoastral array phenotype and the cell cycle arrest in mitosis induced by monastrol are reversible. Celis recover to form a normal bipolar mitotic spindle, to complete mitosis and to proceed through the cell cycle and normal cell proliferation. These data suggest that an inhibitor of Eg5 which induced a transient mitotic arrest may not be effective for the treatment of cancer cell proliferation. Nonetheless, the discovery that monasirol causes mitotic arrest is intriguing and hence there is a need to further study and identify compounds which can be used to modulate the Eg5 motor protein in a manner that would be effective in the treatment of human cancers.

There is also a need to explore the use of these compounds in combination with other antineoplastic agents.

VEGF (also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF can be produced by a wide variety of tissues, and its overexpression or aberrant expression can result in a variety disorders, including cancers and retinal disorders such as age-related macular degeneration and other angiogenic disorders.

Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAI).

WO 99/32619 (Fire ef al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target

RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al; and

WO 99/61631, Heifetz er al), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer ef al). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene,

Summary of the Invention

Disclosed are compositions having two double-stranded ribonucleic acids (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) and a human

VEGF gene in a cell. The dsRNAg are formulated in a stable nucleic acid lipid particle (SNALP). Also disclosed are method for using the composition to decrease expression of

Eg5/KSP and/or VEGF in a cell, and method of treatment of 2 disease, e.g., liver cancer, using the compositions of the invention.

Accordingly, disclosed herein is a composition having a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg3/KSP) gene in a cell and a second dsRNA for inhibiting expression of 2 human VEGF in a cell, wherein both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALP); said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand has a first sequence and said first antisense strand has a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1311

(5 -UCGAGAAUCUAAACUAACU-3"), wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and said second dsRNA. consists of a second sense strand and a second antisense strand, said second sense strand having a third sequence and sald second antisense strand having a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID

NO:1538 (5°-GCACAUAGGAGAGAUGAGCUU-3"}, wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in length. :

In some embodiments, the first antisense strand has a second sequence complementary to SEQ ID NO:1311 (5’-UCGAGAAUCUAAACUAACU-3") and the second antisense strand has a fourth sequence complementary to SEQ ID NO:1538 (5°-

GCACAUAGGAGAGAUGAGCUU-3"). In other embodiments, the first dsRNA consists of a sense strand consisting of SEQ ID NO: 1534 (5’-UCGAGAAUCUAAACUAACUTT-37) and an antisense strand consisting of SEQ ID NO:1535 (5°-

AGUUAGUUUAGAUUCUCGATT-3") and the second dsRNA consists of a sense strand consisting of SEQ ID NO:1536 (5’-GCACAUAGGAGAGAUGAGCUU-3"), and an antisense strand consisting of SEQ ID NO:1537 (5’-AAGCUCAUCUCUCCUAUGUGCUG- 37). In further embodiments, each strand is modified as follows to include a 2’-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate 4s indicated by a lower case letter “s”: the first dsRNA consists of a sense strand consisting of

SEQ ID NO:1240 (5 -ucGAGAAucuAAAcuAAcuTsT-3"), and an antisense strand consisting of SEQ ID NO:1241 (5°-AGUUAGUUUAGAUUCUCGATST); the second dsRNA consists of a sense strand consisting of SEQ ID NO:1242 (5° -GeAcAuAGGAGAGAuGAGCUsU-3) and an antisense strand consisting of SEQ ID NO: 1243 (5°-

AAGCUcAUCUCUCCuAuGuGCusG-37).

In some embodiments, the first dsRNA contains two overhangs and the second dsRNA contains an overhang at the 3° of the antisense and a blunt end at the 5” end of the antisense strand.

The first and second dsRNA can have at least one modified nucleotide. For example, each dsRNA can have at least one modified nucleotide chosen from the group of: a 2'-O- methyl modified nucleotide, a nucleotide having a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. The modified nucleotide can be chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2°-

amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base having nucleotide. In some embodiments, the first and second dsRNA each comprise at least one 2'-O-methyl modified ribonucleotide and at least one nucleotide having a 5'-phosphorothioate group.

Each strand of each dsRNA can be, e.g., 19-23 bases in length, or, alternatively 21-23 bases in length, In one embodiment, each strand of the first dsRNA is 21 bases in length and the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length.

In some embodiments, the first and second dsRNA are present in an equimolar ratio.

As described herein, the dsRNAs are formulated as SNAPS. In some embodiments, the SNALP formulation includes DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA.

For example, the SNALP can have the components in the proportions listed in Table 17,

The composition of the invention can be used to reduce expression of Eg5 and/or

VAGF. In some embodiments, the composition of the invention, upon contact with a cell expressing Eg5, inhibits expression of Eg5 by at least 40, 50, 60, 70, 80, or by at least 90%.

In other embodiments, the composition of the invention, upon contact with a cell expressing

VEGF, inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or by at least 90%.

Administration of the composition to a cell can expression of both Eg5 and VEGF in said cell. The composition of claims I-17, wherein the composition is administered in a nM concentration.

Administration of the composition of the invention to a cell can result in, e.g., an increase in mono-aster formation in the cell. Administration of the composition to a manumal can result in at least one effect selected from the group consisting of prevention of tumor growth, reduction in tumor growth, or prolonged survival in said mammal. The effect can be measured using at least one assay selected from the group consisting of determination of body weight, determination of organ weight, visual inspection, mRNA analysis, serum AFP analysis and survival monitoring. Included are compositions with these effect when adminisiered in a nM concentration.

In a further embodiment the composition of the invention includes Sorafenib,

Also included in the invention are methods of suing the compositions of the invention.

In one embodiment is are methods for inhibiting the expression of Eg5/KSP and VEGF in a cell by administering any of the compositions of the invention to the cell. Other embodiments are methods for preventing tumor growth, reducing tumor growth, or prolonging survival in 2 mammal in need of treatment for cancer by administering the composition to said mammal. In some embodiments the mammal has liver cancer, e.g., the mammal is a human with liver cancer. The method can include a further step of administering Sorafenib.

Brief Description of the Figures

FIG. 1 is a graph showing liver weights as percentage of body weight following administration of SNALP-siRNAs in a Flep3B mouse model.

F1Gs. 2A-2D are graphs showing the effects of SNALP-siRNAs on body weightin a

Hep3B mouse model,

FIG. 3 is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model,

FIG. 4 is a graph showing the body weight in untreated control animals.

FIG. 5 is a graph showing the effects of contro! luciferase-SNALP siRNAs on body weight in a Hep3B mouse model.

FIG. 6 is a graph showing the effects of VSP-SNALP siRNAs on body weight in a

Hep3B mouse model.

FIG. 7A is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 7B is a graph showing the effects of SNALP-siRNAs on serum AFP levels ag measurad by serum ELISA in a Hep3B mouse model

FIG. 8 is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 9 13 a graph showing the effects of SNALP-siIRNAs on human KSP levels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 10 is a graph showing the effects of SNALP-siRNAs on human VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11A is a graph showing the effects of SNALP-siRNAs on mouse VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11B is a set of graphs showing the effects of SNALP-siRNAs on human

GAPDH levels and serum AFP levels in 2a HepdB mouse model.

F1Gs. 12A-12C are graphs showing the effects of SNALP-siRNAs on tumor KSP,

VEGF and GAPDH levels in a Hep3B mouse model.

FIG. 13A and FIG. 13B are graphs showing the effects of SNALP-siRNAs on survival in mice with hepatic tumors. Treatment was started at 18 days (FIG, 13A) and 26 days (FIG. 13B) after tumor cell seeding.

FIG. 14 is a graph showing the effects of SNALP-siRNAs on serum alpha fetoprotein (AFP) levels.

FIG. 15A and 15B are images of H&E stained sections in tumor bearing animals . (three weeks afier Hep3B cell implantation) were administered 2 mg/kg SNALP-VSP (A) or 2 mg/kg SNALP-Luc (B). Twenty four hours later, tumor bearing liver lobes were processed for histological analysis. Arrows indicate mono asters.

FIG. 16 is a flow diagram illustrating the manufacturing process of ALN-VSPDSOI1.

FIG. 17 is a cryo-transmission electron microscope (cryo-TEM) image of ALN-

VSPO2.

FIG. 18 is a flow diagram illustrating the manufacturing process of ALN-VSPG(2.

FIG. 19 is a graph illustrating the effects on survival of administration SNALP formulated siRNA and Sorafenib.

Detailed Description of the Invention

The vention provides compositions and methods for inhibiting the expression of the

Eg5 gene and VEGF gene in a cell or mammal using the dsRNAs. The dsRNAs are preferably packaged in a stable nucleic acid particle (SNALP). The invention also provides compositions and methods for treating pathological conditions and diseases, such as liver cancer, in a mammal caused by the expression of the EgS gene and VEGF genes. The dsRNA directs the sequence-specific degradation of mRNA through a process known as

RNA interference (RNAI),

The following detailed description discloses how to make and use the compositions containing dsRNAs to inhibit the expression of the Eg5 gene and VEGF genes, respectively, as well as compositions and methods for treating diseases and disorders caused by the expression of these genes, such as cancer. The pharmaceutical compositions featured in the invention include a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary 10 at least part of an RNA transcript of the Eg5 gene, together with a pharmaceutically acceptable carrier. The compositions featured in the invention also include a dsRNA having an antisense strand having a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the VEGF gene.

Accordingly, certain aspects of the invention provide pharmaceutical compositions containing the Eg5 and VEGF dsRNAs and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Eg5 gene and the VEGF gene respectively, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Eg5 and VEGF genes.

L Definitions :

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail. "G," "C," "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “d1” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.

For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form

G-U Wobble base pairing with the target mRNA. Sequences comprising such replacement moieties are embodiments of the invention.

As used herein, “Eg5” refers to the human kinesin family member 11, which is also known as KIF11, Eg5, HKSP, KSP, KNSL1 or TRIPS. EgS5 sequence can be found as NCBI

GenelD:3832, HGNC ID: HGNC: 6388 and RefSeq ID number:’NM 004523. The terms “Eg5” and “KSP” and “Eg5/K.SP are used interchangeably

As used herein, VEGF, also mown as vascular permeability factor, is an angiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The VEGF gene contains § exons that express a 189-amino acid protein isoform. A 165-amino acid isoform lacks the residues encoded by exon 6, whereas a 121-amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform predicted to contain 145 amino acids and to lack exon 7, VEGF can act on endothelial cells by binding to an endothelial tyrosine kinase receptor, such as Fit-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is involved in endothelial cell differentiation and vasculogenesis. A third receptor, VEGFR-3, has been implicated in lymphogenesis.

The various isoforms have different biologic activities and clinical implications. For example, VEGF 145 induces angiogenesis and like VEGF189 (but unlike VEGF165)

VEGF143 binds efficiently to the extracellular matrix by a mechanism that is not dependent on extracellular matrix-associated heparin sulfates. VEGF displays activity as an endothelial cell mitogen and chemoattractant in vitro and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a wide variety of cancer cell types and promotes the growth of tumors by inducing the development of tumor-associated vascuiature. Inhibition of VEGF function has been shown to limit both the growth of primary experimental tumors as well as the incidence of metastases in immunocompromised mice. Various dsRNAs directed to

VEGF are described in co-pending US Ser. No. 11/078,073 and 11/340,080, which are hereby incorporated by reference in their entirety.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the EgS/KSP and/or

VEGF gene, including mRNA that is a product of RNA processing of a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and uniess otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, I mM EDTA, 50°C or 70°C for 12-16 hours followed by washing, Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonuciectide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein,

However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity, For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary” for the purposes of the invention. “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, GU Wobble or

Hoogstein base pairing,

The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding Eg5/KSP and/or VEGF) including a 5° UTR, an open reading frame (ORF), or a 3’ UTR. For example, a polynucleotide is complementary to at least a part of a Eg5 mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding Eg5.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a duplex structure comprising two anti-parallel and substantially complementary, as defined above,

nucleic acid strands,. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5 end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”.

Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise ong or more nucleotide overhangs. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucieotides that protrude from the duplex structure of a dsRNA when a 3™-end of one strand : of the dsRNA extends beyond the 5-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA 1s a dsRNA that is double-stranded over its entire length, {.e., no nucieotide overhang at either end of the molecule. In some embodiments the dsRNA can have a nucleotide overhang at one end of the duplex and a blunt end at the other end.

The term “antisense strand” refers to the strand of & dsRNA which includes 2 region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally the most tolerated mismatches are in the terminal regions, e.g., within 6, §, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus.

The term “sense strand,” as used herein, refers to the sirand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand,

As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A

SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iIRNA agent or a plasmid from which an IRNA agent is transcribed. “Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be "introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically, In vitro introduction into a cell includes methods known in the art such as electroporation and

Hpofection.

The terms “silence” and “inhibit the expression of” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to the Eg5 and/or VEGF gene, herein refer to the at least partial suppression of the expression of the Eg5 gene, as manifested by a reduction of the amount of EgS mRNA and/or VEGF mRNA which may be isolated from a first cell or group of cells in which the Eg5 and/or VEGF gene is transcribed and which has or have been treated such that the expression of the EgS and/or VEGF gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of (mRNA in control cells} - (mRNA in treated cells) «100% {mRNA in control cells)

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to EgS and/or VEGF gene expression, e.g. the amount of protein encoded by the Eg5 and/or VEGF gene which is produced by a cell, or the number of cells displaying a certain phenotype, e.g. apoptosis. In principle, target gene silencing can be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of the Eg5 gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of the EgS gene (or VEGF gene) is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 60%, 70%, or 80% ~ by administration of the double-stranded oligonucleotide of the invention. In other embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. The Tables and

Example below provides values for inhibition of expression using various Eg5 and/or VEGF dsRNA molecules at various concentrations.

As used herein in the context of EgS expression (or VEGF expression), the terms “treat”, "treatment", and the like, refer to relief from or alleviation of pathological processes mediated by Eg5 and/or VEGF expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by EgS and/or VEGF expression), the terms "treat", "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, such as the slowing and progression of hepatic carcinoma,

As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by EgS and/or VEGF expression or an overt symptom of pathological processes mediated by Eg5 and/or VEGF expression. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by Eg5 and/or VEGF expression, the patient's history and age, the stage of pathological processes mediated by Eg5 and/or VEGF expression, and the administration of other anti-pathological processes mediated by Eg and/or VEGF expression agents.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. As described in more detail below, such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives, Suitable inert diluents inctude sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. I desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

As used herein, a “ransformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.

IL Double-stranded ribenucleic acid (dsRNA)

As described in more detail below, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 and/or VEGF gene in a cell or mammal, wherein the dsRNA comprises an anfisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the Eg5 and/or VEGF gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said EgS and/or VEGF gene, inhibits the expression of said EgS and/or VEGF gene.

The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

The dsRNA comprises two strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the Eg5 and/or VEGF gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. In other embodiments the duplex structure is 25-30 base pairs in length.

In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ. For example, a composition can include a first dsRNA targeted to Eg5 with a duplex length of 19 base pairs and a second dsRNA targeted to

VEGF with a duplex length of 21 base pairs.

Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. In other embodiments the region of complementarity is 25-30 nucleotides in length,

In one embodiment the region of complementarity is 19 nucleotides in length. In another embodiment the region of complementarity is 21 nucleotides in length. When two different siRNAs are used in combination, the region of complementarity can be identical or can differ. For example, a composition can include a first dsRNA targeted to EgS with a region of complementarity of 19 nucleotides and a second dsRNA targeted to VEGF with a region of complementarity of 21 nucleotides.

Fach strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, or 24 nucleotides in length, In other embodiments, each is strand is 25-30 base pairs in length. Bach strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ. For example, a composition can include a dsRNA targeted to Eg5 with a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides, and a second dsRNA targeted to VEGF with a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.

The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single- stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment,

the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3° end and the 5? end over the sense strand. In further embodiments, the sense strand of the dsRNA has - nucleotides overhangs each at the 3° end and the 5’ end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang 1s located at the 3'-terminal end of the antisense sirand or, alternatively, at the 3*-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5’-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, ie., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3’-end, and the 5°-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate,

As described in more detail herein, the composition of the invention includes a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF. The first and second dsRNA can have the same overhang architecture, e.g., number of nucleotide overhangs on each strand, or each dsRNA can have 2 different architecture. In one embodiment, the first dsRNA targeting Eg5 includes a 2 nucleotide overhang at the 3° end of each strand and the second dsRNA targeting VEGF includes a 2 nucleotide overhang on the 3” end of the antisense strand and a blunt end at the 5° end of the antisense strand (e.g., the 3° end of the sense strand).

In one embodiment, the Eg5 gene targeted by the dsRNA of the invention is the human Eg5 gene. In one embodiment, the antisense strand of the dsRNA targeting Eg5 comprises at least 15 contiguous nucleotides of one of the antisense sequences of Table 1-3.

In specific embodiments, the first sequence of the dsRNA 1s selected from one of the sense strands of Tables 1-3 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1-3. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1-3 can readily be determined using the target sequence and the flanking Eg5 sequence. In some embodiments the dsRNA targeted to Eg5 will comprise at least two nucleotide sequence selected from the groups of sequences provided in

Tables 1-3. One of the two sequences is complementary to the other of the two sequences,

with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of the Eg5 gene. As such, the dsRNA will comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 13 and the second oligonucleotide is described as the antisense strand in Tables 1-3

In embodiments using a second dsRNA targeting VEGF, such agents are exemplified in the Examples, Tables 42 and 4b, and in co-pending US Serial Nos: 11/078,073 and 11/340,080, herein incorporated by reference. In one embodiment the dsRNA targeting

VEGF has an antisense strand complementary to at least 15 contiguous nucleotides of the

VEGF target sequences described in Table 4a. In other embodiments, the dsRNA targeting

VEGF comprises one of the antisense sequences of Table 4b, or one of the sense sequences of Table 4b, or comprises one of the duplexes (sense and antisense strands) of Table 4b.

The skilled person is well aware that dsSRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir er al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1-3, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt, It can be reasonably expected that shorter dsSRNAs comprising one of the sequences of

Tables 1-3 minus only a few nucleotides on one or both ends may be similarly effective as : compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of

Tables 1-3, and differing in their ability to inhibit the expression of the Eg5 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30 % inhibition from a dsRNA comprising the full sequence, are contemplated by the invention, Further dsRNAs that cleave within the target sequence provided in Tables 1-3 can readily be made using the

EgS sequence and the target sequence provided. Additional dsRNA targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending US Serial Nos: 11/078,073 and 11/340,080, herein incorporated by reference.

In addition, the RNA] agents provided in Tables 1-3 identify a site in the EgS mRNA that is susceptible to RNAI based cleavage. As such the present invention further includes

RNAI] agents, e.g., dsRNA, that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNA1 agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent.

Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1-3 coupled to additional nucleotide sequences taken from the region contiguous fo the selected sequence in the EgS gene. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target Eg5 gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1-3. Additional RNA] agents, e.g., dsRNA, targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending US Serial Nos: 11/078,073 and 11/340,080, herein incorporated by reference.

The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5” or 3” end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to aregion of the Eg5 gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides, The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Eg5 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the Eg5 gene is important, especially if the particular region of complementarity in the Eg5 gene is known to have polymorphic sequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhance stability.

The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S.L. er ol. (Edrs.), John Wiley & Sons, Inc, New York, NY, USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2-5" linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are inked 3'-5' to 5-3" or 2-5" to 5'-2", Various salts, mixed salts and free acid forms are also included.

Representative U.S, patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, cach of which is herein incorporated by reference

Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucieoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic intermucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenchydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH? component parts.

Representative U.S. patents that teach the preparation of the above oligonucieosides include, but are not limited to, U.S. Pat. Nos. 5,034,500; 5,166,315; 5,185,444: 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677, 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,566,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein mcorporated by reference.

In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups, The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

Representative U.S. patents that teach the preparation of PNA compounds include, but are not - limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen er al., Science, 1991, 254, 1457-1500.

Most preferred embodiments of the invention are dsSRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular ~CH,--NH--

CH;--, --CH3--N(CH3)--0--CH,--[known as a methylene (methylimino) or MMI backbone], - -CH,--0--N(CH3)--CHy--, --CHa--N(CH3}--N(CH3)--CH,-- and --N(CH3)--CH,--CHj-- [wherein the native phosphodiester backbone is represented as --O--P--0--CH,~] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced

U.S. Pat. No. 5,602,240. Also preferred are dsRN As having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRINAs may alse contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2' position: OH, F; O-, S-, or N-alkyl; O-, S-, or

N-alkenyl; O-, S- or N-atkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C; to Cy alkyl or Cs; to Cy alkenyl and alkynyl.

Particularly preferred are O[(CH2),0],CHs, O(CH1):0CH;, O(CH,)oNH,, O(CH;),CHa,

O(CH)WONH;, and O(CH),ON[(CH3),CHa)l», where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2' position: C; to Co lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, GCN, CJ, Br, CN,

CFi, OCF;, SOCH3, SO,CHz, ONO,, NO,, Ni, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamine, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--

CH.CH,OCHSs;, also known ag 2'-0-{2-methoxyethyl} or 2-MOE) (Martin ef al., Helv. Chim. :

Acta, 1695, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)ON(CH;), group, also known as 2'-

DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy {also known in the art as 2™-O-dimethylaminoethoxyethyl or 2“DMAEOE}, i.e., 2'-O--CH;--0-

CH,--N{CH;),, also described in examples herein below.

Other preferred modifications include 2'-methoxy (2'-OCHs;), 2"-aminopropoxy (2'-

OCH,CH,CH,NH,) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3' position of the sugar on the 3 terminal nucleotide or in 2'-5' linked dsRNAs and the 5’ position of 5' terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 3,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simply as "base™) modifications or substitutions. As used herein, "unmodified" or "nafural® nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- amincadenine, 5-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propyny! uracil and cytosine, 6-azo uracil, cytosine and thymine, S-uracil (pseudouracil), 4-thiouracil, &-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl anal other §-substituted adenines and guanines, 5-halo, particularly S-bromo, 5- triflucromethyl and other 3-substituted uracils and cytosine’s, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3- deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat,

No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And

Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302,

Crooke, S. T. and Lebley, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention, These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracii and 5- propynylcytosine. S-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds,

DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2-0- methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,006; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177, 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which 1s herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

Conjugates

Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger ef al., Proc. Natl, Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan ef al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan er al., Ann. N.Y. Acad. Sci., 1992, 660, 306- 309; Manoharan ef al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholestero} (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras ef al, EMBO J, 1991, 10, 1111-1118; Kabanov ef al,

FEBS Lett., 1990, 259, 327-330; Svinarchuk ef al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac- ghycero-3-Hphosphonate (Manoharan er al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea ef al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan ef al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett, 1993, 36, 3651-3654), a palmityl moiety (Mishra ef al.,

Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino- carbonyloxycholesterol moiety (Crooke ef al., J. Pharmacol. Exp. Ther, 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not Limited to, U.S. Pat. Nos. 4,828,979; 4,048 882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735, i} 4,667,025; 4,762,779; 4,789,737, 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469, 5,258,506; 5,262,536; 5,272,250; 5,292,873, 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585 481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

It is pot necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in 2 single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds, "Chimeric" dsRNA compounds or "chimeras," in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e, a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA: DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can ofien be obtained with shorter dsRNAs when chimeric dsRNA are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region.

Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger er al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett, 1994, 4:1053), a thioether, e.g, hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306;

Manoharan er al., Bioorg. Med. Chem. Let, 1993, 3:2765), a thiocholesterol (Oberhauser ez al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras ef al, EMBO J, 1991, 10:111; Kabanov et al., FEBS Lett, 1990, 259:327: Svinarchuk er al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-gilycero-3-H-phosphonate (Manoharan ef al., Tetrahedron Lett, 1995, 36:3651; Shea ef al. Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan ef al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra ef al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.

Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase.

Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

In some cases, a ligand can be multifunctional and/or a dsRNA can be conjugated to more than one ligand. For example, the dsRNA can be conjugated to one ligand for improved uptake and to a second ligand for improved release.

Vector encoded RNAi agents

In another aspect of the invention, EgS and VEGF specific dsRNA molecules that are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1696), 12:5-10; Skillern, A, et al., International PCT Publication No. WO 00/22113, Conrad, Iniernational PCT Publication No. WO 00/22114, and Conrad, US Pat.

No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a fransgene integrated into the host genome. The transgene can also be constructed io permit it to be inherited as an extrachromosomal plasmid (Gassmann, ef al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by = linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro, Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, ef al., BioTechnigues (1998) 6:615), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld ef al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Blitis, et al, Science (1985) 230:1395-1398; Danos and

Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl.

Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Prac. Natl. Acad, Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury er al., 1991, Science 254:1802- 1805; van Beusechem. ef al., 1992, Proc. Natl. Acad. Sci. US4 89:7640-19 ; Kay et al., 1992,

Human Gene T herapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892- 10895; Hwu er al, 1993, J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent

No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT

Application WO 86/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette ef al., 1991, Human Gene Therapy 2:5-10; Cone et al, 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g, rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector,

Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E er af. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30: and Rubinson D A er al, Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or Il RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H er al. (2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R ef al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R ez al. (1989), J. Virol. 63: 3822-3826; 1.8. Pat, No, 3,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and

International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase IT (e.g. CMV early promoter or actin promoter or Ul snRNA promoter) or generally RNA polymerase ITT promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini ef al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2513)).

In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al.; 1994, FASEB I. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and tsopropyl-beta-D1 -thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Altematively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell. dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsSRNA-mediated knockdowns targeting different regions of a single EGS gene (or VEGF gene) or multiple Eg5 genes (or

VEGF genes) over a period of a week or more are also contemplated by the invention.

Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

The Eg5 specific dsRNA molecules and VEGF specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen er al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Pharmaceutical compositions containing dsRNA

In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a Eg5/KSP and/or

VEGF gene, such as pathological processes mediated by EgS/KSP and/or VEGF expression, e.g, liver cancer. Such pharmaceutical compositions are formulated based on the mode of delivery.

Dosage

The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of EG5/KSP and/or VEGF genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of I to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

The pharmaceutical composition can be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day .

The effect of a single dose on EGS5/KSP AND/OR VEGF levels is long lasting, such that subsequent doses are administered at not more than 7 day intervals, or at not more than 1, 2, 3, or 4 week mtervals.

In some embodiments the dsRNA is administered using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in cach sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly usefinl for delivery of agents at a particular site, such as could be used with the agents of the present invention, In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the : disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vive testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by EG3/KSP AND/OR

VEGFexpression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human EG5/KSP AND/OR VEGF. Another suitable mouse mode] 18 a transgenic mouse carrying a transgene that expresses human EGS/KSP

AND/OR VEGF,

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the

LD30 (the dose lethal to 50% of the population) and the EDS0 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDS0/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention les generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence {e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression, In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Administration

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether Jocal or systemic treatment is desired and upon the area 10 be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, and subdermal, oral or parenteral, e.g., subcutaneous.

Typically, when treating a mammal with hyperlipidemia, the dsRNA molecules are administered systemically via parental means, Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. For example, dsRNAs, conjugated or unconjugate or formulated with or without liposomes, can be administered intravenously to a patient. For such, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases, Such selutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers). Formulations are described in more detail herein.

The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

Fonnulations

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed

Hquids, self-emulsifying solids and self-emulsifying semisolids. In one aspect are forrmulations that target the liver when treating hepatic disorders such as hyperlipidemia.

In addition, dsRNA that target the BG5/KSP AND/OR VEGFgene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the EgS/KSP and/or VEGFgene can contain other therapeutic agents such as other cancer therapeutics or one or more dsRNA compounds that target non-EGS/KSP AND/OR VEGFgenes.

Oral, parenteral, topical, and biologic formulations

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets, Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof,

Suitable bile acids/salts include chenodeoxycholic acid (CDCA} and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, Hnolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1- dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DSRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.

Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly{isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE- hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, - polyhexylacrylate, poty(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Patent 6,887,906, U.S. patent publication. No. 20030027780, and

U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

DsRNAg featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsSRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, capryvlic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl I-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a Cy.io alkyl ester (e.g., isopropylmyristate [PM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference, In addition, dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in virro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including Lipofectin” (Invitrogen/Life

Technologies, Carlsbad, Calif.) and Effectene™ (Qiagen, Valencia, Calif). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton er al. (Nature

Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta ez al, J. Am

Soc. Nephrol. 7: 1728 (1996). Additional information regarding the use of liposomes to deliver nucleic acids can be found in 10.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat Biotechnol. 23(8):1002-7.

Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNA molecules to liver cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.

Characterization of formulated dsRNAs

Formulations prepared by either the in-line mixing or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA).

Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular

Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-

X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For

SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 30 nm.

Liposomal] formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome™ means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall, Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable fo use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,

Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the Hipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when Hposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side- effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high- molecular weight DNA into the skin, Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang ef al.,

Biochem, Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou ef af, Journal of Controlled Release, 1992, 19, 269- 274).

One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral Iiposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoy! phosphatidyicholine (DPPC).

Anionic liposome compositions generally are formed from dimyristoy! phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dicleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylicholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner er al., Journal of Drug Targeting, 1992, 2, 405- 410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/po- lyoxyethylene-10-steary! ether) and Novasome™™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu er al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used “herein, refers to liposomes comprising one or more specialized lipids that, when incorporated nto liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome {A) comprises one or more glycolipids, such as monosialoganglioside Gu, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol {PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized Hpids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen er al., FEBS Letters, 1987, 223, 42; Wu et al,

Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art.

Papahadjopoulos er ¢l. (Ann, NY. Acad. Sci, 1987, 507, 64) reported the ability of monosialoganglioside Gy, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon er al. (Proc,

Natl. Acad. Sci. U.S.A, 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to

Allen ef al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside Gap or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb er al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat- idylcholine are disclosed in WO 67/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto er of, (Bull,

Chem. Scc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C) 21356, that contains a PEG moiety. lum ef al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glvcols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899), Kiibanov et al. (FEBS Lett, 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with

PEG or PEG stearate have significant increases in blood circulation half-lives. Blume ef al.

(Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG- derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound

PEG moieties on their external surface are described in European Patent No. EP (0 445 131

BI and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle er al. (U.S. Pat.

Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European

Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin er al.) and in WO 94/20073 (Zalipsky ef al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi ef al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and {J.8. Pat. No. 5,556,948 (Tagawa ef al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to

Rahman ef al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love er al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition, Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin,

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc, New York,

N.Y., 1988, p. 285). :

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.

Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure, Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric, Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y, 1988, p. 285).

SNALPs

In one embodiment, a dSRNA featured in the invention is fully encapsulated in the lipid formulation to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term "SNALP™ refers to a stable nucleic acid-lipid particle, including SPLP.

As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid

: DNA encapsulated within a Hpid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG- lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they Co exhibit extended circulation lifetimes following intravenous (1.v.)} injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include "pSPLP," which mciude an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 am, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid- lipid particles of the present invention are resistant in aqueous solution to degradation with a nuciease, Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g,

U.S. Patent Nos, 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT

Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium - chioride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N~(I -(2,3- dioleoyloxyipropy]}-N,N,N-trimethylammonium chloride (DOTAP), N-(T -(2,3- dioleyloxy)propy))-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethy1-2,3- dioleyloxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-

Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane {DLinD AP), 1,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linolevloxy-3-dimethylaminopropane {DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt {DLin-TMA.CI), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Ch), 1,2-Dilinoleyloxy-3- (N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino}-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N.N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-

{1,3]-dioxolane (DLin-K-DMA) or analogs thereof, or a mixture thereof. The cati onic hipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle. :

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[ 1 31 dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane is described in United States provisional patent application number 61/107,998 filed on October 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2-Dilinoleyi-4- dimethylaminoethyl-{1,3-dioxclane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidyicholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dicleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoy! phosphatidy! ethanolamine (DPPE), dimyristoyiphosphoethanofamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-cleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a

PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl {Ciz), a

PEG-dimyristyloxypropyl (Cis), a PEG-dipalmityloxypropyl (Cis), or a PEG- distearyloxypropy! (Cs). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

LNPOL

In one embodiment, the lipidoid ND98-4HCI (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid- siRNA nanoparticles (ie, LNPO! particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA {e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid- siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane {e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. © Nec

INN 0 oO NTIS

ND38 lsomer

Formula 1

LNPQ1 formulations are described, e.g., in International Application Publication

No. WO 2008/042973, which is hereby incorporated by reference.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 um in diameter (Idson, in Pharmaceutical

Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,

N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and

Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY., Volume 1, p. 245; Block in

. ~~ Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,

Inc., New York, N.Y, volume 2, p. 335; Higuchi ef a/,, in Remington's Pharmaceutical

Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of cither the water-in-oil (w/o) or the oil-in-water {o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion.

Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water {o/w) emulsion.

Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a soiution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/0) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not.

Multiple enmulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise & system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion, " Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the extemal or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion, Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in

Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,

Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, inn Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel

Dekker, Inc., New York, N.Y, volume I, p. 285; Idson, in Pharmaceutical Dosage Forms,

Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y, 1988, volume

1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion, The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations, Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume I, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions, These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y, volume 1, p. 335; Idson, in Pharmaceutical

Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,

N.Y., volume 1, p. 199}.

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylceliulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts,

benzzlkonium chioride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.

Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in

Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,

Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and

Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y, volume 1, p. 245; Idson, in

Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,

Inc., New York, N.Y, volume 1, p. 199). Mineral-oil base laxatives, oil-sofuble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y, volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M.,

Ed., 1989, VCH Publishers, New York, pages 185-213). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil- in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,

Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has vielded a comprehensive knowledge, to one skilled in the art, of how to "formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and

Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y, volume 1, p. 245; Block, in

Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,

Inc., New York, N.Y, volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not Hmited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polvglycerol fatty acid esters, tetraglycerol monolaurate (M1310), tetraglycerol monooleate (M0310, hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (POS00), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate ~ (SO750), decaglycerol decaoleate (DAQ750), alone or in combination with cosurfactants.

The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butancl, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol,

PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355,

Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides ef al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin.

Pharmacol, 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, case of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides ef al., Pharmaceutical Rescarch, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 83, 138-143). Often microenmuisions may form spontaneous ly when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs.

Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories-- surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee ef al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals.

Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the agneous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemica! emulsions, such as FC-43. Takahashi et al, J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1- monocaprate, 1-dodecylazacycloheptan-2-one, acylicarnitines, acylcholines, Cy.10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, erc.) (Lee ef al, Critical Reviews in

Therapeutic Drug Carryier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic

Drug Carrier Systems, 1990, 7, 1-33; El Hariri ef al, J. Pharm. Pharmacol, 1992, 44, 651- 654),

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's

The Pharmacological Basis of Therapeutics, 9th Ed., Hardman ef al. Bds., McGraw-Hill,

New York, 1996, pp. 934-935). Various natura! bile salts, and their synthetic derivatives, act as penetration enhancers, Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitabie bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glvcodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's

Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1- 33; Yamamoto et al., J. Pharm. Exp. Ther, 1992, 263, 25; Yamashita ef ¢/., J. Pharm. Sci, 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRN As through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr, 1993, 618, 315-339). Suitable chelating agents include but are not

Hmited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, S-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-6 and N-amino acyl derivatives of beta-diketones {(enamines)(Lee ef al., Critical Reviews in

Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Crifical Reviews in

Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur ef al,, J. Control Rel,, 1990, 14, 43- 513.

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in

Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, [-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee er al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 023; and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone {Yamashita ef al, J. Pharm. Pharmacol, 1987, 39, 621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,703,188), cationic glycerol derivatives, and polycationic molecules, such as pelylysine (Lollo ef al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glveols such as ethylene glycol and propylene glycol, pyrrols such as 2- pyrrol, azones, and terpenes such as limonene and menthone.

Carriers dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier” (also referred to herein as an "excipient") is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (Z.e., does not possess biological activity per se) but is recognized as a nucleic acid by ir vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extra- circulatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For exampie, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4"isothiocyano-stilbene- 2,2'-disulfonic acid (Miyao et al., DSRNA Res. Dev, 1995, 5, 115-121; Takakura ef al.,

DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologicaily inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are pot limited to, binding agents {e.g., pregelatinized maize starch, polyvinyipyrrolidone or hydroxypropyl methylcellulose, etc.); fillers {e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, efc.); tubricants (e.g., magnesium stearate, falc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch,

polyethylene glycols, sodium benzoate, sodium acetate, efc.); disintegrants {e.g., starch, sodium starch glycolate, erc.); and wetting agents (e.g., sodium lauryl sulphate, etc). )

Pharmaceutically acceptable organic or inorganic excipients suitable for non- parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcetlulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or sotutions of the nucleic acids in liquid or solid oil bases. The solutions may alse contain buffers, diluents and other suitable additives. Pharmaceuticaily acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels, Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringent, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.

The suspension may also contain stabilizers.

Combination therapy

In one aspect, a composition of the invention can be used in combination therapy.

The term "combination therapy” includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment}. For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention. The compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.

In one aspect of the invention, the subject compounds may be administered in combination with one or more separate agents that modulate protein kinases involved in various discase states. Examples of such kinases may include, but are not limited to: serine/threonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen activated protein kinases (MAPK), meiosis specific kinase (MEK), RAF and aurora kinase, Examples of receptor kinase families include epidermal growth factor receptor (EGFR) (e.g., HER2/neu, HER 3, HER4, ErbB,

ErbB2, BrbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor (e.g.

FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA, SEX); insulin receptor (e.g.

IGFI-R); Eph (e.g. CEKS, CEKR, EBK, ECK, EEK, EHK-I, EEK-2, ELK, EPH, ERK, HFK,

MDK2, MDKS, SEK); AxI (e.g. Mer/Nyk, Rse); RET; and platelet- derived growth factor receptor (PDGFR) (e.g. PDGFu-R, PDGB-R, CSF1-R/FMS, SCF- R/C-KIT, VEGF-R/FLT,

NEK/FLK1, FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but are not limited to, BCR-ABL {e.g. p43™, ARG); BTK (e.g. ITK/EMT, TEC); CSK, FAK, FPS, JAK,

SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may be administered in combination with one or more agents that modulate non-kinase biological targets or processes. Such targets inchide histone deacetylases (HDAC), DNA methyltransferase {DNMT), heat shock proteins (e.g., HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplastic agents (e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibit one or more biological targets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec,

Sutent, Sprycel, Nexavar, Sorafentb, CNF2024, RG108, BMS387032, Affmitak, Avastin, . Herceptin, Erbitux, AG24322, PD325901 , ZD6474, PD 184322, Obatodax, ABT737 and

AEE788. Such combinations may enhance therapeutic efficacy over efficacy achieved by any of the agents alone and may prevent or delay the appearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease for the purposes of shrinking tumors, destroying remaining cancer cells left over after surgery, inducing remission, maintaining remission and/or alleviating symptoms relating to the cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents such as mustard gas derivatives (Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines (thiotepa, hexamethyimelanine), Alkylsulfonates (Busulfan), Hydrazines and Triazines (Altretamine,

Procarbazine, Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine and

Streptozocin), fosfamide and metal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloids such as Podophyllotoxing (Etoposide and Tenisopide), Taxanes (Paclitaxel and

Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine and Vinorelbine), and

Camptothecan analogs (Trinotecan and Topotecan); anti-tumor antibiotics such as

Chromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin,

Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such as folic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, and Gemcitabine), purine antagonists {6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors : (Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and

Pentostatin); topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,

Trastuzumab, hrittmomab Tioxetan, Cetuximab, Panifurnumab, Tositumomab,

Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotide reductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor (Mitotane); enzymes (Asparaginase and

Pegaspargase); anti-microtubule agents (Estramustine); and retinoids (Bexarotene,

Isotretinoin, Tretinoin (ATRA). In certain preferred embodiments, the compounds of the invention are administered in combination with a chemoprotective agent. Chemoprotective agents act to protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane,

In one aspect of the invention, the subject compounds are administered in combination with radiation therapy. Radiation is commonly delivered intemally (implantation : of radioactive material near cancer site) or externally from a machine that employs photon (x- ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

It will be appreciated that compounds of the invention can be used in combination with an immunotherapeutic agent. One form of immunotherapy is the generation of an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a site distant from the tumor. Various types of vaccines have been proposed, including isolated turnor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the subject to be treated, or a derivative of such cells (reviewed by

Schirrmacher er al. (1995) J. Cancer Res. Chin. Oncol. 121 :487). In U.S. Pat. No. 5,484,596,

Hanna Jr. ez al. claim a method for treating a resectable carcinoma to prevent recurrence or metastases, comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of about 107 cells.

It will be appreciated that the compounds of the invention may advantageously be used in conjunction with one or more adiunctive therapeutic agents. Examples of suitable agents for adjunctive therapy include steroids, such as corticosteroids (amcinonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 7-propionate, cortisone, deflazacort, desoximetasone, diffucortolone valerate, dexamethasone, dexamethasone sodium phosphate, desonide, furcate, fluocinonide, fluocinolone acetonide, halecinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, methyl prednisolone, mometasone, prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, and halobetasol proprionate); a SHT1 agonist, such as a triptan (e.g. sumatriptan or naratriptan); an adenosine Al agonist; an EP ligand; an NMDA modulator, such as a glycine antagonist; a sodium channel blocker (e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); a cannabinoid; acetaminophen or phenacetin; a 5 -lipoxygenase inhibitor; a leukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentin and related compounds; a tricyclic antidepressant (e.g. amitryptilline); a neurone stabilizing antiepileptic drug; a mono-aminergic uptake inhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; a nitric oxide synthase (NOS) inhibitor, such as an iNOS or an aENQS inhibitor; an inhibitor of the release, or action, of tumour necrosis factor a; an antibody therapy, such as a monoclonal antibody therapy, an antiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or an immune system modulator (e.g. interferon); an opioid analgesic; a local anaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g. ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g. aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); a decongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, or dextramethorphan}; a diuretic; or a sedating or non-sedating antihistamine.

The compounds of the invention can be co-administered with siRNA that target other genes. For example, a compound of the invention can be co-administered with an siRNA targeted to a c-Myc gene. In one example, AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myc targeted siRNAs are disclosed in United States patent application number 12/373,039 which is herein incorporated by reference.

Methods for treating diseases caused by expression of the Eg5 and VEGF genes

The invention relates in particular to the use of a composition containing at least two dsRNAs, one targeting an Eg5 gene, and one targeting a VEGF gene, for the treatment of a cancer, such ag liver cancer, ¢.g., for inhibiting tumor growth and tumor metastasis. For example, a composition, such as pharmaceutical composition, may be used for the treatment of solid tumors, like intrahepatic tumors such as may occur in cancers of the liver. A composition containing a dsRNA targeting Eg5 and a dsRNA targeting VEGF may also be used to treat other tumors and cancers, such as breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma and for the treatment of skin cancer, like melanoma, for the treatment of lymphomas and blood cancer. The invention further relates to the use of a composition containing an Eg5 dsRNA and a VEGF dsRNA for inhibiting accumulation of ascites fluid and pleural effusion in different types of cancer, e.g., liver cancer, breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer, melanoma, lymphomas and blood cancer. Owing to the inhibitory effects on Eg5 and

VEGF expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

In one embodiment, a patient having a tumor associated with AFP expression, or a tumor secreting AFP, e.g., a hepatoma or teratoma, is treated. In certain embodiments, the patient has a malignant teratoma, an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, a hepatoblastoma, a heptocellular carcinoma, testicular cancer or ovarian cancer,

The invention furthermore relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating cancer or for preventing tumor metastasis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of 2 specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin- dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as "adjunct antineoplastic modalities." Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.

Methods for inhibiting expression of the Eg¢S gene and the VEGF gene

In yet another aspect, the invention provides a method for inhibiting the expression of the Eg5 gene and the VEGF gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target Eg5 gene and the target VEGF gene is silenced.

In one embodiment, a method for inhibiting EgS gene expression and VEGF gene expression includes administering a composition containing two different dsRNA molecules, one having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the Eg5 gene and the other having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the VEGF gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conilict, the present specification, including definitions, will control. In addition, the maierials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Example 1. dsRNA synthesis

Source of reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA synthesis

For screening of dsRNA, single-stranded RNAs were produced by solid phase synthesis on a scale of 1 umole using an Expedite 8909 synthesizer (Applied Biosystems,

Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500A,

Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2'-(-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2-0O-methyl phosphoramidites, respectively (Proligo

Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry,

Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA.

Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%).

Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anion exchange

HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 6408, Beckman Coulter GmbH, UnterschleiBheim,

Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85 - 90°C for 3 minutes and cooled to room temperature over a period of 3 - 4 hours. The annealed RNA solution was stored at -20 °C until use.

Conjugates

The following is a prophetic description of the synthesis of 3’-cholesterol-conjugated siRNAs (herein referred to as -Chol-3"), an appropriately modified solid support was used for

RNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1 4-dicarboxylate AA

G

ANNO

H ©

AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice- cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then,

ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%). 3- {Ethoxycarbonylmethyl-{6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]- amino }-propionic acid ethyl ester AB

Q

AoA Or : FmocHN eg 0

AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0°C. It was then followed by the addition of Diethyl-azabutane-1,4- dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol}. The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water.

The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50 % EtOAC/Hexanes) to yield 11.87 g (88%) of AB. 3-[(6-Amino-hexanoyl}-ethoxycarbonyimethyl-amino}-propionic acid ethyl ester AC

O eta oO

HN XY

AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyi]- amino} -propionic acid ethyl ester AB (11.5 g, 21.3 mmol} was dissolved in 20% piperidine in dimethylformamide at 0°C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexy1)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetradecahydro-} H-cyclopenta{a]phenanthren-3-yloxycarbonylamino]- hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD a

STON

AV he © 0

AD

The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]- propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane, The suspension was cooled to 0°C on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%). 1-{6-{17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetradecahydro-1H-cyclopenta[a] phenanthren-3-yioxycarbonylaminol-hexanoyl}-4-0xo- pyrrolidine-3-carboxylic acid ethy! ester AE 0 oT

O eh

AT

O

AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0°C on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5°C during the addition. The stirring was continued for 30 mins at 0°C and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH,PO4H,0 in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0°C and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%). [6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-0x0-hexyl]-carbamic acid 17- (1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8.9,10,11,12,13,14,15,16,1 7 -tetradecahydro- 1 H- cyclopenta[a]phenanthren-3-yl ester AF

Dati 0 enh

Aor T 0

O

AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b- ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HC] (12.5 mL) was added, the mixture was extracted with ethylacetate (3 x 40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCI;3) (39%). (6-{3-{Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-y1} - 6-ox0-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyD)-10,13-dimethyi- 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro- 1 H-cyclopenta[a]phenanthren-3-y1 ester

AG

OCHs

Oo

AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2 x 5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4’ -dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring, The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with IM agueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2%

MeOH/Chloroform, Rf = 0.5 in 5% MeOH/CHCl3) (1.75 g, 93%).

Succinic acid mono-(4-{bis-(4-methoxy-phenyl)-phenyl-methoxymethyli-1-{6-[17- (1,5-dimethyl-hexyl}-10,13-dimethy] 2,3,4,7,8,9,10,11,12,13,14,15,16,1 7-tetradecahydro-1H cyclopentafalphenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-y1) ester AH

H4CO g : ro 0 CHO ) © C$ OCH; 4B

N

Pi 0 0

AH

Compound AG (1.0 g, 1.05 mmol} was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wi%, 30 mL) and water (2 X 20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.

Cholesterol derivatised CPG Al

H3CO , TOLL oh PB 0 CS OCHj

N

A 0 0

Al

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile {3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2’ -Dithio-bis(5S-nitropyridine)} (0.075 g, 0.242 mmol} in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture tured bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5'-12-dodecanoic acid bisdecylamide group (herein referred to as "5'-C32-") or a 5'-cholesteryl derivative group (herein referred to as "5'-

Chol-") was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the S'-end of the nucleic acid oligomer. dsRNA targeting the Eg5 gene

Initial Screening set siRNA design was carried out to identify siRNAs targeting EgS (also known as

KIF11, HSKP, KNSL1 and TRIPS). Human mRNA sequences to EgS, RefSeq ID number NM 004523, was used.

siRNA duplexes cross-reactive to hurnan and mouse Eg5 were designed. Twenty-four duplexes were synthesized for screening. (Table la). A second screening set was defined with 266 siRNAs targeting human Eg5, as well as its rhesus monkey ortholog (Table 2a). An expanded screening set was selected with 328 siRNA targeting human Eg5, with no necessity to hit any Eg5 mRNA of other species (Table 3a). :

The sequences for human and a partial rhesus EgS mRNAs were downloaded from

NCBI Nucleotide database and the human sequence was further on used as reference sequence (Human BG5:NM_004523.2, 4908 bp, and Rhesus EGS: XM_001087644.1, 878 bp (only 5° part of human EGS)

For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine: u,c-2’-O-methyl nucleotides: s-phosphorothioate linkage.

Table 1a. Sequences of E¢S/ KSP dsRNA dunlexes position in SEQ a oQ ese >. sence of 23mer target w sense sequence {57-37 o antisense sequence {57-37} duplex sequence Ea i. . © or | | oS || Soma || Lm

TRE ON bl I aay a avs | tas | FOTOVRIOSORIR |p | prippieasaniny | gq | pmmasesans | G00 q GUGLT GIsT 757 6246

[Ee [ee [ [a 111 5 Couce uTsT TaT 6247

Table 1b. Analysis of EeS/KSP ds duplexes single dose screen @ nM [% SDs 2nd screen duplex residual {among nama mRNA] gquadruplicates)

AL-DP-6226 23% 3% :

AL-DP-8227 89% 10%

AL-DP-8228 33% 2%

AL-DP-6229 2% 2%

AL-DP-6230 66% 11%

AL-DP-8231 17% 1%

AL-DP-6232 9% 3%

AL-DP-6233 24%, 6%

AL-DP-6234 91% 2%

AL-DP-6235 112% 4%

AL-DP-6236 69% 4%

AL-DP-6237 42% 2%

AL-DP-6238 45% 2%

AL-DP-6238 2% 1%

AL-DP-8240 48% 2%

AL-DP-8241 41% 2%

AL-DP-6242 8% 2% :

AlL-DP-6243 7% 1%

AlL-DP-6244 6% 2%

AL-DP-6245 12% 2%

AL-DP-6246 28% 3%

AL-DP-8247 71% 4%

AL-DP-6248 5% 2%

AL-DP-6248 28% 3%

Table 22. Sequences of Bg3/ KSP dsRNA duplexes ho sequence of i%-mer oo sense sequence {57-3} oH antisenae sequence (5'- Quplex 0: target site No. NO. } nama 268 CAUACUCUAGUCGUUCCCA 49 CRuACuCLAGUCEUNCCCATsT 30 UGGCARCCACUAGRGUAUGT ST AD-12072 260 AGCGCCCAUUCAAUAGUAG BL AGeGoCcCAuuCAABAGUAGTST 52 CudCuAUUCAAUGGECECUTST AD-12073 270 GGAAAGCURGCGCCCRUUC 53 GGAAAGCOUAGCECe chun TsT Ha GAAUGGGCGCUAGCUUUCCTST AD-12074 271 GARAGCURGCGCCCAUUCA BS GRARAGCUAGCGCCCAUUCATST 56 UGRAUGEGCECUAGCUTUCT ST AD-12075 272 AGABRCURCGAUUGAUGGA BY AGAAACUACGRAUUGANGGATST = UCCcAUCRAUCGUAGUUUCUT ST AD-12076 273 UGUUCCUUAUCGAGAAUCT 29 WEIUCCULALCGAGARLC TST 60 AGAUUCUCGRLUARGGAACATST AD-12077 274 CAGAUUACCUCUGCGAGCC 61 cAGALUACCuCuGCGAGESTaT 62 GGCUCGCAGAGGUARUCTIETST AD-12078 275 GCGCCCAUJCAAUAGUAGE 63 GoGoocANUCAAVABUAGATST 64 UCLACUAUUGAAUGGGCGCT sT AD-12079 276 UUGCACUAUCUUTGCEUAT 65 uuGCACubuCcuUGeGUALTST 6g AUACGCAARGRUAGUGCAATST AD-12080 277 CAGAGCGGARAGCURGCGC 67 CAGAGCCCAMAGCUAGCGE TST £8 GCECuAGCUIUCCGCUCTHGETsT AD-12081 278 AGACCUURUUUGGUAAUCT 69 AGAccunhunuGGuAkucuTsT 70 AGATNAC CARALARGGUCUTST AD-12082 279 AUUCUCUUGGAGGGLGUAC 7] AvucucuuGEASGEGCGuUAaTaT 72 GUACGCCCUCCIRGAGARIT ST AD-12083 380 GGCUGGUAUARUTCCACGY 73 GGouGGuAvAALUCCACGUTST 74 ACGUGCGAAIUALVACCAGCCT aT AD-12084 281 GCGGAAAGCUAGCGCCCRY 73 GCGGARAGCUAGCSeccAuTsT 76 AUGEECECUAGCUTUUCCGI TST AD-12085 282 UGSCACUAUCUUUGCGURUG 77 uGchcudncuniGoGuANGTsT 78 CAUACGCARAGALAGUGCATST AD-12086 283 GUAUAAUGCCACGUACCCU 72 GuAvAsuuccAcGuhcooulsT 80 AGGGLUACGUGGARTVAUACTST AD-12087 284 ACARUCUARACURACUAGA B81 AGARUCUBAACUAACUAGAT ST BZ UCuAGUuAGUUUAGAUUCU TST AD-12088 285 AGGAGCUGAAUAGGGUUAC 83 AGGAGCUGARVAGGGUuACT a? 84 CUAACCCLAUUCAGCUCCU TST AD-12089 186 GAASUACATAAGACCUUAT 83 GRAGUACRAURAGACCUGAUT ST Bé& AuAAGCUCUBAUGUACTUCTST AD-12000 287 GACAGUGGUCGAURAGRUA 87 GRCAGUGGOCGRUARGALAT ST 88 UAUCULRAUCGGCCACTUGTCT ST AD-12081 288 ARACCACULGRAGURGUGUCC 89 ARAccAcuuAGUAGUGUEETsT ag GGACRCUACUAAGUGGUUUTST AD-12092 268 UCCCUAGACUUCCCUAUUY G1 UcCCuAGACUNCCoRALRLGUTST 92 AAPUAGGGRAGUCGAGGEATT AD-12093 280 URGACUUCCCURULUCSCU B83 uhGAcvuccouhuaucGouTsT 84 AGCGRARUAGGGARGUT UAT ST AD-12004 291 GCGUCGCAGCCARARDUCGY BE GoGucGohGoohRAUUCGUTST 96 ACGARUUUGSCUGCGACGC TT AD-12095 292 AGCUAGCGCCCAUTCAAUA 7 AZcuAGeGooccAuucAAnATaT 98 VATUGAAUGGGCGIUAGCTTST AD-12096 203 GAAACUACGRUUGAUGGAG 89 GAARCUACGAUCGAUGGAGTST 160 CULcAUCRAUCGUASTUUCT ST AD-12097 204 COGAUARGAUAGAAGAUCA 141 CeGAURAGAUAGARGRAUCATST 1G2 UGRUCTUCWAUCTUANCEET ST AD-12098 208 UAGCGCCCAUUCARUAGER 103 wAGeSocochuucARUAGUATST 104 UACUAUTGEARUIGGGCGC UAT ST AD-12009 296 UUUGCGUAUGGCCARACUG 105 wLuGCGuANGGe CARBCUEGTST Lue CAGUIUGGCCAUACGCARATST AD-12H0 197 CACGUACCCUUCAUCARAL 107 CRCGUACCCUBCAUCABRAUTST 108 AUDDGAUGRAGGGUACGUGT ST AD-12101 298 UCUUUGCGUAUGGCCABAC 109 oUUBGCERAUGEC CARACT ST 110 GUUUGGCCAUBRCGCARAGAT ST AD-12102 200 CCEGAAGUGIUGUTUGUCCA 111 coGARGuGLUSUBLUGLCCATST 112 UGGACARACARR CACUHUCGET ST AD-12103

A000 AGAGCGGRAAGCUAGCGLC 113 ASAGCGGABAGCUAGLGC CTs T 1i4 CLCGCNAGCUUTCCGCUCUTST AD-12104 301 GCUAGCGCCCATUCARUAG 115 GuuAGCEoCCRUUCARAGT ST 116 CUATUGAAUGGGUGCUAGCTST AD-12105 62 AAGUUAGUGUACCGRACUGG 117 BAGUUAGUGUACGARCULGTsT 118 CCABUTCGUACACHRACTUT ST AD-12106 303 GUACGABCUGSAGGAULGG 118 GuACGAACUGGAGEAIUGET ST 120 CCRAGCCUCCAGURCGUACT ST AD-12107 364 ACGRACUGGAGGAUUGGCY 121 AoGRACUGSACGALUGGo LTT 122 BGCCAAUCCUCCAGUTCGIT ST AD-12108 305 AGAUUGAUGUUURACCGRAG 123 AGAuuGhuGuuuccGAAGT aT 1Z24 CUUCSCUARACAUCARICUT ST AD-12108 306 ~~ UAUGGGCUAUAAUUGCACU 123 VARGGECcuAUARUUGCACUTST 126 AGUGCARUUAUAGCCCAUATST AD-12110 307 AUCUUUGCGUAUGGCCAAR 127 AucuuuGcGuAUGECCRAATST 128 UUUGGCCAVACGCRAAGAUTST AD-12111 348 ACUCUTAGUCGUUCCCACUL 129 AcucuAGucGuuccchouctsT 138 GAGUSGGAACGACUAGACTTST AD-12112 309 BACUACGAUUGAUGGAGASA 131 ABRCUACGRUUGAUGGRAGRAATST 132 CUCUCCAGCAATCGUAGUUTST AD-12913 310 GAURRAGAGAGCUCGGGAAG 133 GAUAAGAGAGCLUCEGERAAGT ST 134 CUUCCCGAGCUCUCTuAULTsT AD-12114 311 UUGAGAAUCUARACUAACY 133 ucGAGARUCUARACUARCUT BT 136 AGUUAGUUGAGAUUCUCGATST AD-12115 312 AACUAACUARGARUCCUCCA 137 AbhcuAAcuAGRAuccuccATsT 138 UGGAGGAUUCLASUGAGTIET ST AD-121186 33 GGAUCGURRGARGGCAGUU 138 GGAGCGUARGRAGG CAG TST 14¢C ARCUGCCTUCUuACGAUCCT ST AD-12417 314 AUCGUAAGAAGGCRGUUGA 141 AuCGUAAGARGGCAGUUGATST 142 UehACUSCCUUCURACGAUTST AD-12118 315 AGGCAGUUGACCARCACAR 143 AGGCAGUUGACCAACACARTEST 144 CUGUGUUGGUCARCUGCCUTST AD-12419 316 UGGCCGRUBRGRUAGRAGA 145 uGGCCGAUBACAUAGRAGAT ST l46 UCUUCUAUCTUANC GEC CRT ST AD-12120 317 UCUAAGGARUATRAGUCAACE 147 GCBAAGGALAVAGUCAACATST 148 UGUIGACUANATCCTuAGRT 2 T AD-12121 318 ACURAGCUUAAUUGCUUUC 149 AoubhGouvhhuuGouuucTsT 150 GARAGURAUUAAGCUUAGUTST AD-12122 119 GUOCAGAUCRACCUIURART 151 GoochAGhuchiccuuuAbuTsT i52 ATOARAGEUIGALCUGEGCTST AD-12123 120 UUARUUUGGCAGAGCGGRA 153 vuAALUUGECAGAGCEEAAT ST 154 UUCCGCUCUGCoRAAUMART ET AD-12124 i121 UUATCGAGARUCUAARCTA 155 vuAUCGAGARUCUARACUATST 156 VACUDUAGRUGCUCGALAAT ST AD-12125 22 CUAGCGCCCAUUCARURGY 157 culdGeGescAuucAAuAGuTsT 158 ACUADUGAAUGGGLGCUAGTST AD-12126 323 ARUAGUAGRAUGUGRUCCY 159 ARUAGUAGABLGUGAuCcCuTST 160 AGGAUCACAUTCLUACUAUUTST AD-2127 324 UACGARALGAAGUUAGUGY 161 VALGAARRGAAGULAGUGUTST 162 ACACORACTRICGUUUCGUATST AD-12128 325 AGARRGUUAGUGUACGARCU 163 AGBARGULAGUGUACGAACUTEY 164 AGUICGUACACUAAZTUCUT ST AD-12129 26 ACUARACRGRUUGLTGETUT 163 ACcuRARCAGAUUGALGULILTST 166 ARACATCARAGCUSUGAGTT ST AD-12130 127 CUUUGCGUAUGGCCARALCY 167 cuuuGeGuluGEocabbcuTaT 168 AGUTIGCCCACACGCARAGTST AD-12131 iz8 ARUGAACGAGUAUACCUGEG 168 AALGAAGAGuUAUACCuGEETsT 170 CCCAGGUALACUCTICAUUTST AD-12132 328 AUBAUUCCACGUACCCUTC 171 AvAAvaccAcGuAcecuuc Ta? 172 GARGEGUACSUGGAAULALITST AD-12133 230 ACGUACCCUUCAUCARRUT 173 AcGulcocuuchucAnkunTs? 174 AAUTTHIGAUGARGGGUACEU TST AD-12134 331 CCUACCCUUCAUCARRTTUYT 175 chuAccouuchucAdiuuuTsT 176 AARTFIUGAUGRAGGGUALCE TST AD-12135 132 GUACCCUUCAUCARRAUTUT 177 GuAcccuuchucAiduuuuTae? 178 AAAAIGUGAUGAAGGGUACTST AD-12136 333 ARCUTACUGAURRUGEUAC 178 AfCcuLACUGAUAAMGEUACTST 180 GuACCAUUATCAGUARGUU ST AD-12137 134 UUCAGUCARRGUGUCUCUG 181 uucAGuchARCuGucucuiTaT 182 CAGRCACACUUUGACUGAATST AD-12138 135 CUCUUAAUCCRAUCRUCUGA 183 wucuuAkuccAucAucuGATsT 184 UcAGAUGAUGGAUUAAGARTST AD-12139 336 ACAGUACACARCAAGGAUG 185 AoAGuACACRACAAGCANSTST 186 CAUCCUUGUIGUGUACUGUT ST AD-12140 137 ARGRAACURCGAUUGAUGG 187 ARGAAACUACTAUUGALGGTST 188 CehUcARUCGUAGUUUCTUTST AD-1214%

= sequence Of 18-nmex ie senso sequence (5° +3) ic antisense sequence {5° - duplex 0 target site NO.

Ne, } Rane 138 RAAACUACGAUUGAUGGAGA 189 AnAcuboGAUNGAUGGAGAT ST 100 UCUCCAUCRRUCGRAGITITST AD-12142 338 UGGAGCUGUUGAUAAGAGA 191 BEGAGCUGUUGARAAGAGATST 182 UCUCUUAUCAACAGCUCCATST AD-12143 340 CURACUAGRAUCCUCCAGG 193 cubRCuAGAAUCCUCCAGGT 87 194 CCUGGAGGRUTCUAGTUAGTST Al-12144 341 GRAUAUGCUCAUAGAGCAR 195 GARuduGoucAuAGAGCEATST 186 UUGCUCUAUGAGCAVADUC TST Al-12145 342 AUGCUCAUAGAGCAAAGAD 197 AuGcucAuASAGCARAGART ST 198 UUCHUUSCUCLAUGAGCALTST AD-12148 343 AAMRAUUGCUGCUGUUGAG 199 ARAAALUGGUGCUGUUGAGTST 200 CUCAAGAGGACCRAAUUUUUTsT AD-12947 344 GAGGAGCUGAAUAGGGUUA 20% GAGGAGCUGAAGAGGGUUATET 202 WAACCCuAUT cAGCTCCUC TT AD-12148 345 CGAGCUGARIAGGGUIACA 203 GEAGCUGARAUAGGGUUACATST 204 UGUAACCCUATICAGCUCC TT AD-12149 344 GAGCUGAAUAGGGUUACAG 205 GAGCuGRARUAGGGUUACAGTT 206 CUGUAACCCORATUCAGCUC TST AD-12150 347 AGCUGARURGGGUUACAGA 207 AGCUGAAUAGGGUUACAGATST 208 UCUGUARCCCUAUUCAGCUT ST AD-12151 348 GCUGAAUAGGGUUACAGAG 208 GeuGAALAGGGUUACAGAGT aT 210 CUCUGUAACCCRATT CAG TST AD-12152 34% CCARACUGSAUCGUARGAR 211 CeARACUGGALCGUARGART 5T 212 JUCUuACGAICCAGUUUGGETST AD-12153 350 CAUCGBUARGAASGCAGUUG 213 CAVCGUARGARAGGCAGUUGT ST 214 CARCUGLCUUCIuACSAUC TT AD-12154 351 ACCUUAUUUGGUARICUGC 21% AccunluuuCGubiucuGeTsT 216 GCAGAULACCARIIAAGGUT ST AD-12155 352 HUAGAUACCAUTACUACAG 217 SUAGAGACCARUACUACAG TST Zig CUCUuAGUAALIGGUALCLART ST AD-12156 353 AUACCAUUACUACRGUAGC 218 AOACCAUACUACAGUAGCTST 220 GCUACUGUAGUAAUGGR ATE T AD-12157 ’ 154 UACUACAGUAGCACULGGA 221 VACGACAGVAGCACURGEAT ST 222 UCCARCUGCUACUSUAGUATST AD-12158 155 LAAGUARAMCUGUACURCAE 223 ARAGUAAAACUGURACUACAT ST 224 UGUAGUACAGUUUUACTUUUTST AD-1215% 356 CUCARGACUGRUCUUCUAR 225 CucARGACUGAULUUCUAATST 226 UuAGRAGAUCAGUCUUGAG TST AD-12160 157 UGGACAGUGGCCGAUARGA 227 VUGACEGUGGECCGAVARGAT ST 228 UCUuAUCGGRCCACUGUCAATST AD-12181 358 UGACAGUGGCCGAURAGAL 228 wGACAGUGGCCGAUAAGAN TT 230 AUCUuATDCGGCCACIGUCATET AD-12162 150 GCAAUGUGGARACCUAACU 231 GeARUGUGGAAARCCURRCUTST 232 AGUUAGGUUUCCACAUUGCTST AD-12163 360 CCROUUAGUAGUGIUCCAGG 233 coAcuuAGUAGUGUCCAGG Ts? 234 CCUGGACACUACUARGUGETST AD-12164 361 AGAAGGUACARRAUUGCUU 235 AGAAGGUACARRRUUGGUU TST 236 AACCARAUGUUUGUACCUUCT TST AD-12165 362 UGGUUUGACURRGCUTRALD 237 UGGEURUGACUAAGCULARLTET 238 AULAAGCULWACTCARACCATST AD-12166 363 GEUUUGACURAGUUUARUU 239 GGUUBGACUAAGouEAALGTST z40 ARUUAAGCUUAGUCAARCITST AD-12167 364 UCUAAGUCAAGAGCCRUCY 241 NCRAAGUCARAGAGCcAucuTsT 242 AGRUCGGCTCUUGACTACAT ST AD12168 36h UCAUCCCURUAGUUCACUYT 243 ucAucoouAvAGuucAouuTsT 244 ALGUGAACURCAGGGAUGATST AlD-12169 360 CAUCCCUAUASUUCACUTT 245 chucoouAuAGuuchcuunTsT 246 RARGUGAACUAGAGGGRUGT ST AD-12170 367 CCCUAGACUUCCCUAUUNC 247 cecuAGACuLCCcuAGuUCTaT 248 GARAANAGGGAAGUCUAGSETST AD-12171 368 AGARCUUCCCUAUTUCECUU 249 AGAcuucceuAuuueGeuaTsT 256 RAGCGAARGAGEGAAGUCUT SY AD-12172 369 UCACCABACCAUUUGUAGR 251 VWCACCARRCCALULGUAGATST 252 UCUACARAUGGUUUGGUGAT ST AL-12173 370 UCCUUUAAGAGGCCURACT 253 UCouNABGAGGoCcuRACUT ST 254 AGUGAGGCCUCUUARAGGATST AD12174 371 UUUAAGAGGCCUMACUCAY 25% VUUAAGAGGCCuAACuCAUTST 256 AGCASUuAGGCCUCTuARAAT aT AD-12175 372 UUAAGAGGCCUARCUCADD 257 vLeAAGARGEecuAAcucAunTsT 258 BAUGAGUuAGGCCUZULAATST AD-12176 373 SGCCUARCUTADUCACCCY 258 GEcouAACucAUUCATCoUTST 280 AGGGUGAAUGAGUUAGGCCT ST AD-12177 374 UGGUAUUTIUGAUCUGECH 261 ugGuAuIULGAUCUGGCATST 262 UGCeAGAUCARARALACCATET AD-12178 375 AGUUUAGUGIGUARAGUUT 2463 AGuuuAGuGuGUAAAGUUUTST 264 AARRCUTUACACACUARECUT ST AD-12179 376 GCCAMMIUCGUCUGCGARG 265 GeoRAARUCGUCUGEGAAGTST 266 CUUCGCAGASEAAUUUGGCS TST AD-12180 377 AAUUCGUCUGCGARGRAGA 267 ARunCSuCuGCGAAGARGATST 268 UCUICUTCGCRAGACCGAATUTST AD-12181 378 UGRALAGGUCACCURAUGAR 260 UGAARGGUCACCUARLGAATST 270 UUCATuAGGUGACCUIU CAT ST AD-12182 379 CAGACCAUUCUAAUUUGGCR 271 chAGAcchouLARIUUCGGCATST 272 UGCCRAATUARADGGUCTE eT AD-12183 380 AGACCAUUUAAUUUSGCAG 273 AGACCAUUUABLULGECAGTST 274 CUGCCAAAUMBARUGGUCUTST AD-12184 381 AGUUAUUAUGGGLUAUAAU 275 AGuvhAuuAUGEGouABARL TET 276 ATuAUAGCCCAuBALARCUTST AD-12185 382 GCUGGUAUBAULCCACGUA 277 GouGGUALARULCIROGLATST 278 UACGUSGARUUAGACCRGCTsT AD-12186 183 AUTRTAAUDUGGCAGAGCGG 279 AUGUUAANGLEGCRGAGLGGTET 28¢ CCGCUCUGCoABAUGARRUTST AD-12187 3g4 UTUAAUUUGGCAGAGCEGA 281 un AAUURGGCAGAGEGGATST 282 JCCECUCUGCcABATLARAT ST AD-12188 185 TUUGGECAGAGCGGARAGTE 283 wu GGECAGAGCGGRAAGSUTST 284 AGUUUTCCECUCUGT cRAAATST AD-12188 386 UUUUACAAUGGRAGGUGAS 2685 UUUUACARUGERAGEUGART ST 266 UUcACCUUCcAUUGUARBATET AD-12180 87 AMUGGBAGGUGARAGGUCE 287 ARUGGAAGGUGRRAGGECATST 288 UGACCUTUCACCUNC CAGUTET AD-12181 389 UGAGAUGCAGACCAUUURA 289 WGAGRuGCAGAccAuuuAA TST 2940 UuAAATGEUCCGCAUCUCATST AD-12182 380 UCGCAGCCABAUUCGUCUG 291 ueGeAGCEARRLLCGUCUGTST 292 cAGACGRAUUUGGCUGCGATST AD-12183 390 GGCUAUTARTUGCACUAUCTU 293 GEcukuiaunGehAculucuTs? 294 ACANAGUGCAATUAGAGCCTST AlD-12194 i101 AUUGACAGUGGCCGAUAAG 233 AuuGACAGUEGCCGRULAAGTST 296 CUUAUCSGCCACUGU CART ST AD-12185 192 CUAGACTUCCCURUTUCEC 287 cuAGAcuuccouAuuueGaTsT z298 GCGARAVAGGEARGUCUAGTST AD-12166 23 ACUAUCUUUGCGUAUGEEC 299 AouvhucuuuGoGuAuGGeeTsT 300 GOCoAuACCCARAGALAGUTST AD-12197 304 AUACUCTAGUCGUUCCCAL 301 AuAcucuAGucGuuccchaTeT 302 GUGGGARCGATUAGAGUALTET AD-12198 385 ARRGAAARCURCGAUFUGAUG 303 AABCGARACUACGALUGAUGT ST 304 CAUCRAICERAGUUUCHULT ST AD-12199 396 GUCHUGATUIUIIGGCGEE 305 GeounGhuuuuuuGGeGGETsT 306 CCCACCABRARANTCAAGGL TST AD-12200 387 CGCCCAUUCARUAGURGAR 307 cGoccAuLcARBAGUACAAT ST 308 JUCLACCAUTGRAUGGGUGTST AD-122014 398 CCUUAUUUGGURBADCUGLY 309 couuAuuuGiurihucuiouTsT 310 AGCACGAUNRCCARRGARAGCT ST AD-12202 1898 AGAGACAATUCCGGAUSUS 311 AGAGACARALUCCEGARGUGTST 312 CACAUCCGGRAUUGUCUCUTST Al-12203 100 UGACUUUGAUAGCUAARUT 313 uGAcuuuGAcAGeuAihhuuTsT 314 AAUTuAGCUAUCBRRGU CATS? AD-12204 10% UGGCAGAGCGGAARGCUAG 315 UEGCAGAGCHGRAAGCUAGT ET 316 CuAGCUUICCECICUGC CATT AD-12205 102 GAGCGGARAAGCUAGCGCCC 317 GRGCGGRAAGCLAGCCceTaT 318 GEGCECUAGCUUUCCGCUCTaT AD-12206 103 AAARGEAGUUAGUGUACGAR 319 ARAGAAGUUAGUGUACCAATST 320 UTCEUACACURACUUCIUUT ST AD-12207 104 AUUGCACUAUCUUUGCGUZR 321 AUUGCACUAUCUNUGCGUATST 322 UACGCARRGAUAGUGCAAUTST AD-12208 105 GGUAUAARUUCCACGUACCC 323 GGuAuAiuucchcCuicec?sT 324 GEGUACGUGGARUINAUACC TST AD-12200 106 UACUCURGUCGUUCCCACT 325 uAcucudGucGuucechcuTsT 326 AGUGGGRACGACVAGAGUATST AD-12210 107 URUGAARGARACUACGAUU 327 VAUGARAGARACUACGAUUTST 3z4 AAUCGUAGUULCUTUCRUATST AD-12211 108 AUGCUAGARMSUACAUALGE 328 AuGCubEAAGUACAUAAGATST 33¢ UCUuAUSUACTUCHAGCATTST AD-12212 10¢ AAGUACAURAGACCUUAUU 331 ARGUACBLARAGACCUUAUUTST 332 AAUAAGGUCUUAUGUACTITET AD-12213 ne sequence of 18-mex we sense sequence (5° -3°) he antisense, sequence (57 - duplex

Oo: target site Ne. NE. 37 Tame 110 ACAGCCUGRGCUGIIARUG 333 ACAGCCUGAGCUGUUARUGTST 334 CAUUARACAGCUCRSGCUGUTST AD-12214 111 AARGRAGAGACAAUUCCGE 335 ARAGAAGAGACRRLUCCGETST 336 CCBGAAUTUGICUCUUCUUETST AD-12215 112 CACACUGGAGAGGUCUAAR 337 cACACUGGAGAGGUCUARATST 338 JU:AGACCUCUCCAGUGUET ST AD-12216 113 CACUGGAGAGGUCUAARGU 3349 CACUSGAGAGGUSUARAGUTST 340 ACUTUAGACCUCUCCAGUGTST AR-12217 314 ACUGGAGAGGUCUARAGUG 341 AcuGGAGAGGUCUARAGUG TST 342 CACUTUAGACCUCUCCAGUTST AD-12218 115 COUCGCAGCCAAAUTUCGUL 343 cGucGeAGecchraunstucTsT 344 GACGRAUUUGGCUGCGACGTST AD-12219 116 GLAGGCAGUUGACCAACRL 345 GRAAGGCACUUGACCARCAC TST 34a GUGUIGGUCARCUSCCUUCTsT AD-12220 117 CAUUCACCCUGACAGAGUT 347 chuuchoccuGACAGAGUUTST 348 AACUCUGUCAGGGUGABUGT ST AD-12221 118 ARGAGECCURACUCAUUCR 346 AAGAGGCCuARCUCALUCATET 350 UGARUGAGUuAGGCIUCTUTST AD-12222 119 GAGACAAGUCCGGRUGUGG 351 GASACAAGCCEGAUGUEETST 352 CCACRUCCEGARTGTGUCTC TST AD-12223 120 UUCCECADGUGEAUGUAGA 353 VUCCEGAUGUGGAUGUAGATST 354 UCuACAUCCACAUCCGGARATST AD-12224 121 BAGCUABCGCCCAUUCAAL 355 AaGcubGoGoocAuncAAn Ts 356 AUUGAAUGGGCECUAGCTUT aT AD-12225 122 GRAGUUAGUGUACGAALCUS 357 GRAGUUAGUGUACGAACUGTST 358 CAGUUCGUACACUAACTUTCTsT AD-12226 123 UAUAAUUCCRCGUACCCUU 355 vAuhAvucchcGuAcccuuTsT 360 AAGGCUACGUGGAAUUALATST AD-12227 124 ACAGUGGCCGAUARGAURG 361 ACAGUGGCoGAVARGRUAGT ST 362 CuAUCTUAUCGGCeACUGUT ST AD-12228 125 UCUGUCAUCCCUATAGUUC 363 ucuSuchucocududGuuetsT 364 GARCUMUAGEGAUGACRGATST AD-12229 126 UUCUUGCUAUGACUOGUGU 365 wacunGeuAuGAcuUNGUGUTsT 366 ACACARSUCAGAGCARGAATST AD-12230 127 GUAAGABGGCRGUUGACCA 367% GUAAGARGGoAGUUGAC CATT 368 UGGUCABCUGCCUUCUUACT ST AD-12231 128 CRUIGRCAGUGCCCGAUAR 369 cAULGACAGUGECCEAUAATSET 370 TuAUCGECCACTUGUAATGT 8 T AD-12232 129 AGARACCACUUAGSUAGUGT 371 AGARACCACUBABUAGUGUTET 372 ACACUACUAAGUGGEUUGCUTET AD-12233 130 GOAUUGUUCAUCARUUGGC 373 GOAULGULCAUCAAUGGCTST 374 GCcARAUUGAUGARCAALCCTST AD-12234 133 UARGAGGCCUAACUCATUUC 375 WAAGAGGeoukBEcucAuucTaT 376 GAAUGAGUUAGGCCUCUATST AD-12235 132 AGIJAGUGUACGARCUGGAE 377 AGuuAZUGEACGRACUGGATST 378 UCCAGTUCGUACACURALCUT ST AD-12238 133 AGUACATARGACCUTTAUTU 378 ASuAcRUBAGACCcUUALLLTST 380 ARRUBAGGUCUAUGUACUTST AD-12237 134 UGAGCCUUBUGUATAGAUT 381 UGAGCcuuGuGUALAGAURTST 382 ARJCUAGACACARGGCUCATET AD-12238 i358 CCUUUAACGAGGCCUARCUC 383 CcouLUAAGAGGCcuAACu CTE T 384 CAGUUAGECCUCUUARAGET ST AD-12239 136 ACCACUUAGUAGUGUCCAG 385 AccAcuuABuAGuGuCcAGTsT 386 CUGGACACUACTVALRGUGRUTST AD-12240 137 GAAACUUCCARUUAUGUCU 387 GabhcuncchhuuAuGueuTsT 388 AGACAUAATUGGRAGUIICT ST AD-12241 £38 UGCAUACUCUAGUCSUUCC 389 uGehuACuCUAGUCGUUCS TST 3%0 GERRCGRACUAGAGUATGCATET AD-12242 $39 AGRAGGCAGUUGACCARCA 361 AGARGECcABuUGACCRACAT ST 362 UEUUSEUcAACUGCCUTUCU TST AD-12243 140 GUACAUAAGACCUUAUTUE 383 GuACAUMAGACCULALUUGT ST 394 CABAGARGGUCUUAUGUACTST AD-12244 141 UADARAUUGCACTAUCTUUS 386 nAudbvuGchovhucuuuETsT 396 CARAGALAGUGoRATuAUATST AD-12245 142 UCUCUGUUACAAUACRUAU 387 ucecubuuhchinAchuduTs? 388 RURUGUAUUGUARCAGAGATST AD-12246 143 UAUGCUCAUAGAGCABBGA 309 GAuGCUCAUAGAGCAAAGATST 400 UCUUUGCUCUAUGAGGAUATST AD-12247 144 UGUUGUUIUGUCCAAUUCUG 401 uGuuGuuuGucchivucuGTay 402 CAGAAUUGGACABACARCATET AD-12248 145 ACURBCUAGRRUCCUCCAG £03 ACuRBCUAGARRCCUCCAGTST A04 CUGGAGGATUCuAGTLAGUTST AD-12249 146 DGUGGUGUCURUACUGARR 405 UGUGGUGUCUALACUGARAA TST 456 GUCCAGUABGAGACACCACATST AD-12250 147 GAUUBUGGGAGRCCACCCA 4G7 VA LAGGEGRGACCACCCATST 408 UGGHUGGUCUCCoAuARGATST AD-12251 148 ALGGAUGARGUCURUCAAR 409 BAGGAuGAACuCRRAUCARA TST £10 YUUGHuAGACUUCAUCCUCT ST AD-122502 148 UUGAUARGAGACCUCGGGA 411 GUEAUAAGAGAGOLUCGEGATST 432 UCCCGAGCUCTCUuAUCAAT ST AD-12253 i580 AUGUUCCUUAUCGAGARIC 413 AuaGuucouuhucGAGAAL Cl sT 414 GAUUCUCGAUBAGGERCRTT ST AD-12254 151 GGAAUAUGCUCAUAGRGCA 415 GRAM AUGCUCAUAGAGCATST 416 UGCUCuAUGAGCAURAUTCS TT AD-12255 152 CCAUUCCRARCUGGAUCGT 417 CCAUUCCARRCUGGRUCGUTST 418 ACSAUCCRGUUUGGRAUGGTST AD-12256 153 GGCAGUUGACCAACACART 419 GGcAGUUGACCAACACARLTSY 420 AUUGUGUUGGLCARCUSGCCTST AD-12257 154 CRUGCUBGRAGUACAUAAG 421 CANGCUAGRAGUACAUAACTST 422 CULAUGuACUTCLAGCAUGTST AD-12258 i55 CUAGAAGUACAUARGACCU 4273 CUAGRAGuACAGARAGAC CUTS? 424 AGEUCUQACGLUACUTCuAST ST AD-12259 156 UUGGAUCUCUCACAUCUAT 429 GLGGAUCuCUCACALCUAUTST 426 AuAGAUBUGAGAGAUCCARTST AD-12260 357 AACUGUGGUGUCUAUACUG 427 ARcuGuitusucudnacutGlsTt 428 CAGUAMAGACACCACAGUUTST AD-12261 158 UCAUUGACAGUGGCCGAURE 429 ucAuuGAGAGUGGCCGAUATST 430 UAUCGGCCACUGUCAAUGATST AD-12262 156 AUBAAGCAGACCCAUUCCC 431 RuArAGCoAGACcocAuuceeTsT 432 GGGARUGGGUCUGCUTUAL TET AD-12263 160 ACAGRAACCACUUAGUAGU 433 ACAGRARCCACUUAGUAGUTST 434 ACGACUAAGUSCUTTGCIGEUTST AD-12264 jel GAAACCACUUAGUAGUGUC 435 GhbhcoacuuAGUAGUGuCTeT 436 CACACUACUAAGUGGUUUCTST AD-12265 162 BAAAUCUGAAGGAURUAGUCA 437 ABAVCUAAGGALAUAGUCATST 438 UGACLAUADCCUUAGRUUUTST AD-12266 1E3 UUAUUUGAUACCCAUCAACA 439 vuhuuuAvAcccAUCAACATST £40 UEUUCAUGGGUALARRLAATST AD-12267 164 ACAGAGGCRUUAACACACU 441 ACBRGAGSCcAuuhACACACUT eT 442 AGUGUCURARUGCCUCUGUEsT AD-12268 1685 ACRCACUGGAGAGSUCUAA 443 ACACACUGGAGAGGLCUAATST £44 CuAGACCUCUCCAGUGUGUTST AD-12269 166 ACARCUSGAGAGGUCUARAG 445 AcAcuGEAGAGGuCuABAGE TST 446 CUUUAGACCUCTCCAGUGUTST AD-12270 167 CGAGCCCAGAUCRACCUUU 447 GGAGCoeAGhuCAACouLL TAT 448 ABAGGUUGAUCUGEECUCETST AD-12271 166 UCCCUADUUCGCUTUCUCE 44% uccouRuuucGouuucucsTsT 450 GGAGRARGCGRABUAGGGATST AD-12272 169 UCUARARUCACUGUCRACE 4B] ueudbAhucAcuGucAACATST 452 COUIGACACUCAUTGuAGAT ST AD-12273 174% LAGCCARATUCGUCUGCGRR 453 AGccArbuucGucuGeGRATST £54 UUCECAGACGRATTUGGECUTST AD-12274 171 CCCAUUCRAUAGUAGRAUG 458 cooBuuchARuAGUAGARWET aT 456 SAUTCARCUBUUGALUGEET ST AD-12275 172 GAUGRAUGCRUACUCUAGU 457 GRuGhRAuGCAuACuCUAGUTeT 458 ACUAGAGUATGCAUUCATICT ST AD-12276 373 CUCROGUUCCULAUCGAGA a59 cucBuGuuCCuuANCGAGATST 460 UCUCGAUAAGGARALBIGAGTST AD22TT 174 GAGAAUCUARACUAACUAG 461 GAGARLCuARLCUARCUAE TST 462 CUuAGUUAGUUUAGAST CCT eT AD-12278 175 UAGRAGUACAUAAGACCUU 463 uAGAAGUACRURAGACCUUTST £64 RAGGUCUGAUGUACUUCUATST AD-12278 178 CAGCCUGAGCUGUULRUGR 465 caGeoubAGoubuuAiAuGAT ST 466 UeAUuARcAGCUCAGGCUGT ST AD-12280 177 AAGAAGAGACAAUUCCGGE 467 BAGAAGAGACARUUCCGGATST 463 UCCEGRAUUGHCTCUUCUT TS T AD-12281 $78 UGCUGCUCUGCAUDGUUCA 469 uGcuGGututGiuuiuuchlsT 470 UGAACARUCCACACCAGCAT:ET AD-12282 176 ARAUUCHUCUGCGARGEAG 471 ARAUNCGUGUGCGAAGAAGTST 472 CUUCUUCGCAGACSARDUUTST AD-12283 180 UUGCUGGAARGUUGAGAUGU 473 WIMCUGGAAGUBGAGAUGUTST 474 ACRUCUCAACUUCCAGRART ST AD-12284 181 TACUARRCAGAUUCGAUGUHT 475 CACUARACAGALUGALGULTST 476 ARCAUCARUCTGUGUAGUAT ST AD-12285 o sequence of Lo-mex Ey sense seguencs {57-37 ES autiosnoe sequence (3'- duplex

Oo target site ¥0. NG. } name 182 GRUUGAUGUUUACCGAAGU 477 GRULGAUGUUUACCGARGUT ST 478 ACUUCGEURAACAUCARUCTST AL-12288 183 GCACUAUCUUUGCGUAUGS 479 GoAcuAucunuGeGuauGETsT 460 CChuACGCAAAGAVAGUGC TET AD-12287 184 UGGUATAATICCACGUACT 481 uGGuALAAaCCAcGrAccTsT 482 GGLACGUGGARTUAUACCAT AT AD-12288 185 AGCAAGCUGCUURACACAG 483 AGCAASCUBCUVARCACAGTST 482 CUGUGUUARGCAGCOUGCUT ST AD-12289 186 CRGARACCACUUAGUAGUG LER CAGAAACCAOUUAGUAGUGTET 486 CACUACUAAGUGEIULCUGTST AD-12280 } 187 AACUUAUUGGAGGUUGUAR 487 ARcuuALUBGAGGUUGUAATET 488 Jub chAlCUCCARLARGUUTST AD-12284 188 CUGGAGAGCUCUARAGUGSE 489 SUGCAGAGGUCUARAGUGE TST 490 CeACUUCABACCHCUCCAGT ET AD-122682 1806 RAAAANGRUAURAGSGCAGU 481 AAAARAGAUBIARGECACGUTET 482 ACUGCCUuALATCOUITIUUT ST AD-12283 180 GRAUUUUGRUAUCUACCCH 493 GARLLLUGAUATCUACCCRATET 464 UGGEUAGAUAUCARRADTCT ST AD-12204 181 GUAUTUTUGAUCUGSCAAC 495 GubvuuuaGAacuGGeAACTST 456 CUUGC CAGAUCARARAVACTST AD-12295 i192 AGERAUCCCUUGGCUGGUAL 427 AGGAuCccuuGEouGGUANTST 458 AUAC CAG CAAGGEAUCTITST AD-12208 £83 GGAUCCCUUGECUGGUATR 49% GGAuccouulBGouBiuAnATsT 500 LGAVACCAGCCAAGGEGAUCCTT AD-12267 $94 CAMIAGUAGARUGUGAUCC 301 CRAUAGUAGARALGUGALRCC TST 502 GGAUCACAUUCUACUAUTCTST AD-12298 i395 GCUAURAUUGCACURUCTUTG 503 GoulAuhbvuGohcubucuuTsT 504 AAGAUASUGCAAUGAUAGCT ST AD-12299 186 UACCCUUCRUCAARGUTUU 505 cAcccuucAuchAiuuuunTsT E06 AARDAUUGUGAUGRAASGGUATST AD-12300 197 AGBACAURUIGAATARGCT HUY AGAACAUVAUUGRAVAAGLCTST 508 SOCTRACUCAAUAUGUUCUT ST AD-12301 ig8 AAAUUGGUGCUGUHUGAGGA 509 ARRUSGuUGCUGULGAGGAT sT 510 UCCUCARCAGCACCRAUUT 'Y AD-12302 £99 UGARUAGBGUUACAGRGUU 511 UGAAUAGEGUUACAGAGUUTST 512 ARCUCTUGUAACCCuBRUUCATST AD-12303 300 AAGAACUUSARACCACUCA 513 AARGRACUUGRAACCACUCATST 514 UGAGUGGUCUCARGUUCUUEST AD-12304 551 AAUABAGUAGARCCCARUUCC 516 ARUBRAGCAGACCCAULCOTST 516 GGEAAUGRGUCUGCUTLAUTTST AD-12305 352 AUACCCAUCARCACUGGUA B17 AubcccAushAcAcuGGuATsT 518 CROCAGUGUUGAUGGOUALT ST AD-12306 533 UGGAUUGUUCALCARUUGE 519 GEGARULGULCAUCERALUGGT ST 520 CchAUDUGAUGAACARUCCAT ST AD-12307 04 UGCAGAGGUCUARAGUGER B21 BGGAGAGGUCNRARCUGGATST 522 UCCACUCuAGACCUCUCCAT ST AD-12308 5305 GUCAUCCCURUAGUUCACU 523 GuchugccuAiuAGuueiouT aT 524 ASUGRACUAUAGECATGAL Ta T AD-12309 5406 AURAUGGCUAUBAUUUCUC 323 AuhhuGGoudvhbusucu cal 326 GAGARAUGRUAGCCATLAUTST AD-12310 307 AUCCCUNGECUGGUALARU 527 AuccounGoubGtGuivalbulsT 528 ATJuAuACCAGUCAAGGEAUTST AD-12311 508 GGGCUAUAAUUGCACUAUC 529 GGGouAuAAUGCACLALCTET 530 GAULASUSCARTUROAGCOC TST AD-12312 508 GAUUCUDCUUGGAGEGCGIRE 531 GhuucucuGEAGEGUGUATST 532 BACGCCCUCCRAGAGARTCT ST AD-12313 5140 GCAUCUCUCARUCUUGAGG 533 GeRucu Cu CRAUCUUGRGGETST £34 COUCAAGAUUGRGAGALGCTST AD-12314 3tl CAGCAGRARAUCUAAGGRTA 535 CAGCAGRERLCUAAGGAUATST 536 VAUCCULAGAUUUCUGCUGT ST AD-12315 512 GUCARAGAGCCAUCUGUAGA 537 GuchAGAGCCAuCuGLAGATST 538 UCLACAGAUGGCUCUUGACT TT AD-12316 513 AMACAGAGECARUUBACACE B30 ARARCAGASCCcAuUAACACATST 540 UGUSUuAAUGCSUCUSUULT ST AD-12317 314 AGCOCAGAUCRACCUUUAR D4L AGcochlhuchAcocuauAATsT 542 DuAAAGGUUGAUCUGGGCUT ET AD-12318 515 DAUTUTUSRICUGGCARALE 543 vAvuuunGAucLEGeAAccTsT a4 GGUUGCCAGAU CABAARDAT ST AD-12319 316 UGUUUGGAGCAUCUARCUAR 543 UEUUIGEAGCAUCUACUAATT 546 TuAGUAGAUGCUCCRARCATST AD-12320 517 GARAUUACARGUACACAACA 547 GHAMIVACAGUACACRACATST 548 UGUUGUGRACUGLARTUTCT ST AD-12321 518 ACUUGACCAGUGUARAICT 549 AcuuGhocASuGuibhucuTsT 550 AGAUTIACACUSGUCARGUTST AD-12322 318 ACCAGUGUARAUCUGRCCU 5531 AccAGUGEARBLCUGACCUTST 552 AGGUeAGAUTUACACHGED TST AD-12323 520 AGAACARUCAUUAGCAGCA 553 AGRARCAAUCHUVAGCACGCATST 554 UGCUGCUARUGATUGULCUTST AD-12324 521 CAAUGUGGAAACCUAACUG B55 CAALGUGEARACCUARCUGT ST 556 CAGUUAGEIUUCCACAUUGT aT AD-12325 522 ACCARGRAGGUAZARAATT 557 ACCRAGRAGGuACARLALLT ST 558 AAGIUTGuACCUUCUTGCU TST AD-12328 523 GOUACARAAUUGSUUGAASZ BED CouheARAAUGGUUGRRAGT ST 560 CUUCBACCAAUTTUGLACCTST AD-12327 524 GGUGUGEAUUGUUCAUCAR 361 GGEUGUSGALLGUUCAUCARTST 562 UUGAUGARCARAUCCACACLT eT AD-12328 325 AGAGUUCACHRARMAGUCCH 563 AGARGUUCACAARARGCCCATST 584 UGGECUUTIUGUGARCTECUT ST AD-12329 526 UGATAGCURAAUUARACCR SE5 wGAGAGCuARALGARRCCATST 5g6 UGGUUAAUUGAGCUAL CATT AD-12330 327 LAUARGCCUGCARGUGARIC 567 BANAAGCoouGRAGUGARANCTST 568 GAUUCACUUCAGGCURAGUTST AD-12331 328 CAGUUGACCARACACARUGE 560 ChGuuGACCARCACARUGCTST 570 GoAUUGUGUUGGUCBACUGT ST AD-12332 528 UGGUGUGGRBUSUUCAUCA 271 wGEUGuEEAvuGuuchucATsT 592 UGAUGAACRAUCCACACCAT ST AD-12333 330 AUUCRCCCUGACAGAREUUC 573 AnucAcCuGACAGAGuueTT 574 GRACUCUGUCAGECUGAAIT aT AD-12334 531 URAGACCUTAUUUGSUART 575 WAAGACCLURAVUVEGOUAR LT ST 576 AUUACCARARRAGGUCTUATST AD-12335 532 AAGCRAUGUGGARACCUAA 577 AAGCAAUGUECARALCUARATST 578 UuhGGUHUCCACAUUGCUITeT AD-12336 533 UCUGARARCUGGAUAUCCCA 579 ucuGhiicuGGAvincocATaY 580 UGSGAuAUCCAGUIUCAGAT 5ST AL-12337

Table 2b. Analysis of Eg5/KSP dsRNA duplexes

I lst single 2nd single dose dose . Ard .

Egh/ ESP screen f Shs ist screen screen B sDs 2nd screen slngie Shs 3rd screen duplex a (EON ” {among dose {among name 50 mo [% guadruplicates) £5 ni [% quadruplicates) screen uadru licat ) rasudual resudual Eh inte a oe oe gquadruplicates mRNA] MRNA]

AD-12072 B5h | eT pe hp

AD-IZ073 | eae le ee Te

ADU2074_ | Sly a | se | ey 1] : ADA2075 | Bey 1 asf 3és 0 4w Lp

ADAZOT6 re Taw 1 ame

Aozora Te ee [iy TTT

AD-12078 | ezw | 3% LBs les |b 0]

AD1Z079

AD-12080 | ees 48 1 oosaw | as bj

AD-12081 | "34% |- ey [35% of 0 eas ff 0]

ADZ0B2 Ov ITE es oe

AD-12083 | hme | ew Teme img

AD12084 | tee | ey | tre | ae {

ADA2086 | iss [ax | aps | ay |]

AD-12086_ | ze I oes has Fv 0

ADzoer | EESTI as [wos | a

AD-12088 I

AD-12089 5% I

A200 | Aew | ase | sax | my

AD-12091 | aes Ges 1 aw | ss 1

AD-t2082 | EER TTT hed ess | my

AD12003 | mew | Ek oe igs

ADizose | ase | sy ms Toy

AD-2005 [14% | ew fp Ass 0 as lb

ADSIZ006 | gee dig he fy

AD1306T [EI TT ew | aie oe

AD72098 | Ais | iaw [in | a

ADA2080 | ene [a ams |e

ADAzio0 | HERE TTT Tad [eee me

AD-zIol [dee [de aa | ah

ADfZi0z | dex | ify mew | ew TTT

AD-12103 soy {0 ose op eee Noa

ADiZIGs [abv | ee hes TTR

ADZ105 | 35% 7 Sev wey 1 —

ADAZI06 | Bib | ee | qe | qos [4

AD-12107

AD-12108

ADIOS | des we ag ge

AD2110_| eee | se | ets | as

AD-A211_ | eae 1 ev 1 vas aes 0

Az [TAR TTT ae Tas ee

AD12113 13%

AD-121%4 | see | ss oo aes fay nf

ADAZ ee | aw ese 1

AD-12116 | 74%

AD-12117 ETT as Ges aw

ADipiE | sae asa Ee

AD-T2110 | ve as has se

ADA2120 | aoe [ex | ey ay

ADAzizd I~ see tie fee 4% bp]

ADE TT TRe wes [dee my

ADA2123_|___ 26% x ie | ooo

AD-izi2e | hey |p hee

ADAZIZS | Ange a

AD 2125 Bem ih

AD-12127 SIR ee Tm my

AD-12126 me eae

ADZIpe | are me ene

ADazia0 | se ev | ans | ov

ADA2131 | aee [sw pmo ae

ADAZIZz | he ae TTT Guy gy

AD-Z13s | aw [Aw ges [ey

ADAZISA | ley me ia am

AD-12185 lst aingle Znd single | 1rd

Eg5/ KSP dose SDs 1st screen dose 50s 2nd screen ; single Sha 3rd screen i. screen @ screen § = oo ore duplex . {among i (among dose {amang

TAME 50 ni {% cuadruplicates) IN guedruplicates) SCrean quadruplicates) reguduzl = resudual - @ os nd AES Rem mRNA] mRNA) wo

ADEE | ann Tew ome ay

AD-2137 | met | ew [ems | gs

AD-12438 | die | es [ees fas [Tm

ADz130 | Bbs | es | ee | ay

Aboioiao [TE TTT ee [ee ee]

ADZ1A1 20nd TT on fips Te

ADf2taz | see | we wn ge 4

AD-12743 | eax 1 sdy [0 ves [0 2% 0 Tf

AD-12144 | see 1 sey) wes fee 8]

ADTIZ1AE ve [es | ae |e

ADA sere ae

ADAZIAT |S nye ee

AD2748 | 30s | 0 se | 0 ses | 00 se 0]

AD-12149 | 8 foes 0 ies] sw 0 [0]

ADAZISO | aie | en | aw om

AD-218T 186 [Re a a

AD-12152 | TR Tam [aT

ADT2153 | 20% ew sis Te

AC-12164 | Edy [7% 0 Cb aes Fs rm Tm

AD2166 | a3 en Tem any

AD I2186 | a ab yy

ADA 2157 Be TTT TTT ame aT

ADAZIsE | mv | ew epee

AD-12169 | mv | er aes | es

AD-T2160_| av |_| ms | gy

ADAZ161 BRT TT en qe)

AD-12157 Pet I EE

Tote fm ae]

ABAZIGE | yp

ADA 2Tes | TEE TTR TT a pe © TADames [se [uo TTT TTR

ADazier | mer | sw [aoe [oy | 1

AD-1268 | 54x fas | sss 1 wee 1 FT]

AD-12169 aly Fas 1 swe loses 1

AD-12170 am anew sey

AD-I21TY | ere law | amy

AD-12172 |S [TTR TT yy

AD-12173 1 ses [os 0 0 dew fee oT

RD-f2t74 aly | ee ow [gy

AD-12175 © zew 4 ov 1 58 laa

RDARTTE Ae | see am

AD-12177 | ess 0 ee fay 0 ste 1

AD-12178 41% 4% ose J es oT

AD-t2179 | 5% | se 0} 4a | 0 ss 1}

ADCiZi80 | aee Ee | is |e

ABATE |e aa a TTT

AD12182 | ew | aw aE ee

AD-Zi83 | ee | sw wee ae

AD-iZtBA | haw | mp am | py

AD2i8s 1 ew 1 aw es hw boo]

ADEs |e lee a ew

AD-13187 | Ses TTT

AD-12188 | sow | a meas

AD-12186 | si | asf ass 0} 000 se i 1 7

AD-12190 33%

AD-12191 20% 2% la Vow i 4

AD-d2if2 | ey AT aw 0 0 ess 0 aos oT od

AD-12193 | eay p00 ws 0 fees [ew 0 TF

AD-12194 1 8s 1 os fp isy [0 ay 1 1

Abies [ain Tse [a

AD-12196

AD2i7 | ee | aw me

ACcioios | se mae [py

AD-12199 © 303% J ew 0b ies bee 0 v0 bo]

AD2200 se | ew gov ia 1

ADOT | Es mie a lst siagle 2nd single avd

BEgS/ ¥SP dose SDs ist screen dose Shs 2nd screen single SDs 3rd screen screen @ - screen B ' JH — SEE dupiex (among . (among dose {among name 50 nit [% gquadruplicates) 25 mM {% gquadruplicates) screen gquadruplicates) resudual : rasudual a a5 a “ rRNA] MRNA]

RDAZeea | dow {aw | ew | am |__

AD-iz20s | ate ign mes [des 1]

AD-taa04 | ere 7a [ees | gy TTT

ADT2205 | ber age [sms [as

AD-12206 dey | sw [am | aes |]

AD-12207 gv 1 sw 4 aos | ew 7

AD-iz2os | abe hs amy [ey

AD-12208 lot Tees | gm

AD-TZo10 | aEs aw a hay

ADAZ2 | des Ter [ioe ey rp

ADAZp77 sae | ose of oesv 1 0 se U1 oo]

AD-12213 Gay Lo es 1 asx | ee Lf]

Azza | en aw [we | ia

AD-12215 Zw ew ame hy

AD-12216 S| an im TTT

AD-a217 1 3s oo [ame ob ew 1

AD-12318 | 35% oes lave bse Vo ot]

AD-12219

ADAza0 av Reem | a

AD-T2z2t seme Sew eg

Abgz Ts ee 1 Es sy

AD-12223 | ey 1 aoe | ers 1 0 9s 0 1]

ADAZise | pee pss | a

AD-iz2zs | ee | se Tee ig

AD-T2226 | ds ew EER

AD-12227 Gy ee Taney

AD-tazas | Ese lm oe | ey

AD-12229

AD-12230 Et

ADZeH TERR lime ms es

AD-12232 | 30% _ {03% 1 divs bo es 0

ADdz23a see LTR ams

AD-z234 | sew | ey eer

ADamss [En | Tam mT

AD2236 | nm yee ga

ACAZ |r ev we ey

AD-236 | io 5 ees es

ADA2230 | ap | se aos | ew

ADA2240 | ane | ev mew | ee

AD-12241 1 69% 0 sy [vos | 000 se | % 0]

ADZEAr ee | ew an | ws

ADizas [Teer | a | gee uw

AD-12244 | pss | es 1 dase 4 aw Fo 1

ADTz2ds [een | my gm | my

RD2288 | 20h | wma ee

Dazed | Ere ase | se | mw [

AD-12248

AD-12249 44% 34%

ADA2350

AD-12251

AD12252

AD-12253

AD-12254

AD-12265 163% 10%

AD2256 | 1:7 £0

AG-12267 10s

AD-12258 2%

AD-12259 20% 5%

AL-12260

AD-12261 VED is 6k

AD-12262

AD-12263

ADiZo64 vm ew wos [iw | won| ax

ADA2265

AD-12266

AD12267 lst single 2nd single dose dase 3rd

Egb/ KSP Shs let screen SDs 2nd screen single She 3rd screen screen 8 screen @ duplex 50 ni (8 {among 25 nM 1% ] (ameng dose {artong name resudual guadrupliicates) resudual quadruplicates) screen quadruplicatces} mRNA] mRNA) 25 nM

AD-12268 34%

AD-12269 | oat

AD-12270 £2

AD-12271 53% ih

AD-12572 | i

AD-12273

AD-12274

AD-12275 | 25%

ARA2276 1 45%

AD-12277

AD12278 130%

AD-12278

AD-p280 | 2% | oe pose Fis ff

AD-12261 z8 ok En ee

AD2285 | 3% 1 owl oo@se ew 4b]

AD2283 1mm aw aes gp

ADAZBE TET ase mT

AD-12285 | 7% | vs 0) iw Geek Lo}

AD-12286

Ab-izasy | ave me | se | gm

AD-12288 | Ze 7% pases 0 tdes 0

ADAzo8s | heme mow | gms

DAZ mn i ene pm TTT

Afzal | _ ew yew

Azer | ew mw ema

Rbze9s | dv aw sen mo opp Tee Le Te

AD-12295 at Ee Tey

Romzas | ENT eer | pe

Apazeay | ie | ese [am

ADazes | Tse | hes [mT

AD=izage | rev | es ees faa TTT

AD-12300 | ses | ae 0 iss V0 ie Tm 1

ADAZOT | ese Tes ape aw op

AD-12302 | ees | ss fess} 00 ss 1 0 T ]

AD12308 { 35% bes FV i7y on es vf]

AD-i2304 | 06 mw |v ie

AD-12305 | en |e Em

AD-12306 23% | oes | eos | 0 se [0 FC

AD-i2307 | ee [es ees | ET

AD-12308 ; zie Tes iss [0 es fb]

AD-I2305 | ee [ae ape | my

AD-12310 306s | whee

ADizal ise | lew | egy | gy

ADMayz mers [wes [se

ADAZ3Is ede es gee | ey

AD-12314 Ee LTR aa ee

ADAZats | aie [Tam ge TTT

AGI2316 | is sy | rey Rp

ADAz3i7 | _sen |e | asy 4 es

AD2518

AD-12319 Te TTT TR se pa

ADazsa0 | Em | we Taw wm

AD-12321 zis | 0003s 0 op aos 1 es lb]

Abmzagz |e | me eyo

ADZa23 des |e [ses | ex 1

ADA Teas ee

ADIZa26 | ae hy Emmy

AD-12326 Ee em | ase aie

AD-123g7 [| 36s 1 ass | seb sey bp

AD-12328 | as% ov of sie + 3% ob 4

ADA2320 | Ele ae Tea wm

ROT2330 | ese | ev mR TTT

Aba | __e0y | ov Ge [my —

AD-12332 | Bow | as 0 oosws J vs [0 1]

AD-12333 | 34% | "6s of sew fey of 7]

1st single 2nd single _ doses dose . 2rd

Eg5/ KSP screen @ SDs lst screen sorecn SDs 2nd screen single SDs 3rd screen duplex 50 nM [% {among 55 no [% . (among dase (among name resudual guadruplicates) resudual quadruplicates) screen quadruplicates)

RRNA] mRNA} 25 nM

AD-12334 | o7e | os Coase 0 ss 000 [000

AD-28% else [aby

AD-i2336 | ese as [ae [mm

ADizaar | aes 1s [des | oy TT

Table 3. Sequences and analysis of EgS/KSP dsRNA duplexes : SDs single Bnd

SEQ . - SEQ gose screen o Cm Antisense sequence (57 - duplex soresn 8

Sense sequence (5'-37) in oo ID {among

NO. 50 NO. name 25 nH [% quadra rggicual 2

RSA] plicat eg) cehunbcuAsAGuAGeAcuTaT E32 AGUGCUACUGUAGUARTIGET ST 583 AD-14085 19% 1% 2ucuGGehhcchuAunucuTsT 584 AGARRVAUGGUIUGCCAGAUTST 588 AD-14086 38% 1%

GRAUAGCUAARUUABACCAATST 586 GUGGUUuAATULAGCUAUCT 5T 87 AD-14087 75% 10%

AGRuAcchunhcuhchGuATsT EEL VACTCUAGUARUGGUAUCTT ST 589 AD-14088 22% 8%

GRuuGuucAuchhueGGoGTaT 550 CGCCAAUUGRUGRACARUICT 8T 581 AD-14089 70% 12%

GouuutueCucGEouchenTsT 582 AGUGAGCCGAGGAGARAGCT ST 583 AD-14000 78% 11%

GEAGGANMGGCRSACRAGAT sT 594 UCULGRICAGCCRAUCCUCCTsT 585 AD-14091 29% 3% uBAUGARGAGUAUACCUGGET ST 596 CoAGGUAUACUCTUCAURATST 587 AD-14092 23% 2% wuuchccAARCCAUUUGUATST 598 WACARAUGGUUUGGUGAARTST 555 AD-14083 0% 2% cuuAuuAAGGAGUAVACGEETAT e00 CCGUANACUCCUuARUAAGT ST 601 AD- 14084 11% 3%

GAAAUCAGAUGGRCGUAAGT ET 662 CUIACGUC cAUCUGAUTUCTsT G6C3 AD-14085 10% 2% cAGAUGUCAGCAUARGCGAT ST 604 UCGCULAUGCUGACADCIGD eT 60h AD-14096 27% 2%

AucuhbccculfuuGuiucTsT 6086 GAGACRACUAGGEUUAGAIIT ST ev AD-14007 45% 6%

AAGAGCUUGUUAARAUCGGTST £08 COGARTTMARCAAGCUCTUT ST 609 AD-14008 50% P0%

UUAAGGAGUAUACBGAGGAT ST 610 UCCUCCGEUAGACUCCTIUAAT ST 611 AD-14099 12% 4% uuGchluGUAAALACGUALT ST 612 ALACGUATUUACAUUGCART ET 613 AD-14100 45% T% ucudhccouAGuuGuduccTsT 614d GOALACAACUAGGEGUUAGATST 615 AD-14101 36% 1% cAuGuauguuuuucucGAnTsT 616 AUCGAGAAAAAGAUACRUGTST G17 AD-14102 49% 3%

GAUGUCAGCAUBAGCGAUGT ST 618 CAUCGCUUADGCUGACAUCT 8 T 51¢ AD-14103 74% 5% uCcecAACAGEUACGACRea TST 620 GGUGUCCLACCUGUUGGEAT ST 621 AD-14104 2% 3% uGCuCACGAUGAGUUVAGUTST 622 ACUAARCUCAUCGUGAGCAT ST 623 AD-14105 34% 4%

AGAGCBUGUIAARRRCCEATST 624 UCLGAUUURARCARGCUCUTST 625 AD-14106 jE 2%

GeGURACABGHACAUCLAUATST 628 VAGAGAUGUUCUUGUACGL TT 627 AD-14107 2% 1%

GAGGuuGuAlGeoAALGURTST 628 BRACAUGCGCTuACARCCUCT 8T 629 AD-14108 15% 1%

RACAGEUACGACACCACAGTST £30 CUGUGGUGUCGLACCUGUUT aT 63% AD-14409 91% 2%

BAccouaSuuduAuccousTaT 632 GAGGEANACARCHAGGEUU TST 633 AD-14110 E6% 5%

GCAVAAGCGAUGGAUARUAT ST £34 pATuAUCCAUCGCUUALUGCT ST 635 AD-141411 33% 3%

BAGOCAGGCEAVARUACCLATST 636 VAGGUAULAUCCAUCGCTUT ST 637 AD-14112 51% 3%

UCZALUCCUGUACGARARAGAATST 638 UUCUUUUCGuACAGGAU CATT 639 AD-14113 22% 3%

BABRACAVUGGCoGUUCUGETST 540 CCAGRACGGCCAAUGUUTUT ST 64% AD-14114 117% 8%

CUUGGACGGGCGUACARGART ST 642 UUCTUGUACECCCUL CARGT ST 643 AD-14115 50% o%

GGoGACRAGARCAUCUAYTT 644 RUAGAUGUUCUUGUACGCCT SY 645 AD-14118 14% 3%

AcucuGhAGuhcAuuGEAAaT ST 646 AUTUCCBAUGUACTCAGAGTTET 547 AD-14117 12% 4%

LuAULAARGGRGUALACGGAT8T 648 UCCGRANACUCCUHUAALART ST 649 AD-14118 26% 4% uAAGGAGUAUACGGAGGAGT ST 8580 CULCUCCOuALACUCCUwAT sT G51 AD-14119 24% 5%

ABRAGCARUGAGUCARCUARAT ST 652 FUuAGUUSACUATTGATTUT 5 T 653 AD-14120 a% 1%

AAucAAUAGUCARCUARDG TAT £54 CUUAGUTUGRCUAUTGAUT 8T 658 AD-14121 24% 2% vucucAGuRUACuGuUEUAATST 656 TuACACAGUAVACTGAGRAT ST 657 AD-14122 10% 1%

LEUGAAL CAC CuGAVARAT ST 658 TUuAUCAGAGUGUUUCACATST 658 AD-14123 8% 1%

AGRUGUGRAUCUCUCARCAT ST [a UGUUCAGAGAUTCACAUCTUT ST 661 AD-14124 O% 2%

LGGuuGuAAGCcAALGUIUGT ET 562 CARCAUTIGGCUUACRACCUTsT 663 AD-14125 114% 5%

UGAGARAUCAGAUGGACEHUTET G64 ACGUCCAUCUGATTUCUCATAT gon AlD-14126 9% 1%

AGARAUCAGAUGCACGUAATST 666 DUACGUCCAUCUGAUTUCT TET 667 AD-14127 57% 0%

AuPucocAACAGBuACGACTST 663 GUCCUACCUGUUGEEAURTET ST 668 AD-14128 104% 0%

CCCARCAGGuAoGACACCATST 670 UGGUGUCGuACCUGTUUCGET aT ait AD-14129 21% 2%

AGuAnkcuGhAAGARAccucuTseT 672 AGAGGUUCUTCAGUAUACTT ST 673 AD-14130 57% 6%

AuAUANBUCAGCCGGLCGC TET 674 GOGBCCCEGCUCAvAVAUAUT ST 615 AD-14131 83% 6%

ARrucurhccouhGuuGuAuTsT e76 AUACARACUAGGGUUAGAUUTEY 877 AD-14132 75% 2% cublcccukGuuGuiucceeTsT 678 GGGALACARCUAGGGULAGT ST 676 AD-14133 60% 4% cuAGuuGuAvcecuccunuTsT 680 AAAGGAGGGAVACBACUAGTST 681 AD-14134 44% 6%

AGAcRuCuGhcudAuGGcuTsT 682 AGCCATTUAGUCAGAUGUCUT ET 683 AD-14135 55% &%

GAAGouCACARUEAUUUAATST 684 TJUARRAUCLUUGUGAGCIUUCT aT 685 AD-14136 28% 3%

AchuGuhucuguuucucGATaT ebe CCEAGARAAAGRUACAUGUTST 687 AD-14137 40% 3% poGAuucARAucunkkoocTsT 668 GEGUWARGATIUCSAAUCSAT aT £89 Al-14138 319% 5%

single §Ds a or dose 2nd ya SEQ Antisense sequence (3° - SEQ duplex screen # aereen

Sense sequence {(H°-37} in 37) ib nam 25 0M [% {among

NO. NG. flame ad rn quadru residual Io

NENA] mllcat es} vouudheccuuAGGAcucuTsT 6890 AGACQUCCUAAGGOUUAACGAT2T SE AD-14138 Ti% 11%

GouchcGAUGAGUUUAGUGT ST 692 CACUARACUCAUCGUGAGDT 8T 603 AD-14140 43% 15% chUAAGCGRAUGGAUABLACT ST 894 GUATWAUCCAUCGCUUAUGT ST GO5 AD-14141 33% 6%

AUAAGCGAUGGAUAALACCT ST 686 GOLATURUCCAUCGCUUAUT ST gov AD-14142 51% 4% conhAuAARCUGeocucAGT ST £98 CUGAGGGCAGUUATUAGET ST 650 AD-14143 42% 1%

HCOGRARGIUGAACIUGGUT ST 700 BCcARGUUCAACTUUCCEATST 701 AD-14144 4% 4%

GhAArRcAuLEEooGuucuGTsT 702 CRGAACGGCCAAUGUUUUCT 8 T 703 AD-14145 82% 5%

ARAGACuGAUSUUCUALGUUT aT 704 AACUUAGARGRUCAGUCUUT sT 7052 AD-14145 13% 2%

GaGcruGuurrbRcGGRAT ST 706 SUCCGAUUUUAACAAGCUCT ST 7467 AD-14147 B% 1%

AcAnuGGoouuouGGaGeTsT 708 GCUCCAGAACGGCehAUGUT ST 7459 AD-14148 B0% 7%

AAGARCcAuCcuRuARUGGoAT ST 710 UG oAMRARAGAUGHUG CUI TST ii AD-14149 44% TE

AARuGuGucuAcuchuGuuTaT 712 AACAUGAGUAGACACAUUUTST 713 AD-14150 32% 20% uGucuAcuchuGuuucucATaT T14 UGAGABACAUGAGURGACATST TLE Al-14151 75% 11%

GuRuhcuGuGuAAchAucuTsT 716 AGRUUGUUACACAGUAMACT ST TL AD-14152 8% 5% .

GAGACUGUGUAACARuCUAT ST 718 VAGAUUGTUACACAGRRURT &T 719 AD-14153 17% 15%

CuuAGUAGUGUCCAGGARAT ST 120 UUUCCUGGACACUACUAACGT 8T re AD-14154 16% 4%

UCAGRUGGACGUANCECAGT ST 122 CUGCCUuACSUCCAUCTIGAT 8T 723 AD-14155 11% 1%

AGAUARAUUGAUAGCACRAATST 724 UUGUGCuAUCRAUTwAUCUT 8T T28 AD-14158 10% 1%

CAACAGGUACGACACCACATST 726 UGUGGUGUCGUACCUGUUET 8T 727 AD-14157 29% 3% uGCAAUGUARAUACGUA LUT ET 728 AAVACGUATULACAUUGCATST 729 AD-14158 51% 3%

AGUCAGARLMUGARCUAGAT ST 730 UCUAGAVARRATUCUGACUTST 731 AD-14159 53% 5% cuhAChAARuCTIUUUAACECC TT 732 SGUGUUARARGAUUUCUAGT ST 733 AD-14160 40% 3%

AbuARAUCuUARCCCUAGUUT ET 734 AACUAGGEUUAGAUTUAUUTsT 735 AD-14161 53% 7%

AAduutucuGoucAcGhuGAT eT 7348 ToAUCCUGAGnAGAARATUT ST 737 AD-14162 44% 6%

GoocucABuARIUCCARGET ST 738 CoAUGGATTUACUGAGEGCT ST Tie AD-14183 57% 3%

AcGuuulAlAicBAGiucuulsT 740 AAGAUCUCSUUTRARACGU TST 741 AD-14164 4% 1%

AGGAGAURACGAACGUURAAAT ST 742 CUuARACGUUCuAUCUCCUT eT 743 AD-14185 11% i%

GACLGUCAUGGELGUCGCAGT ST 744 CUGCGACGCCAUGACGEUCY eT 745 AD-14166 90% 5%

AcoGuCALGERGUCGCAGeT TY 746 GCUGCGACGCCAUGACGGUT 8T 747 AD-14167 49% 1%

GARCGUUUARMARCGAGRUCTST T48 GAUCUCGUUURARACGUUCTST 749 Al-14168 12% 2% puUGAGCUL ARC AURGEUAATST 750 UuACCUAUGUUARGCUCART 87 751 AD-14169 66% 4%

ZoudArivuGhocucGuAGATST TH2 UCuACGAGAUCARTURAGTT ST 753 AD-141T0 52% 6% uecGuAGARULANCURAAUATST 754 wATUAAGANAATUCUACGAT ST 755 AD-14171 42% 4%

GEAGALAGARCGUUVAARART ST 756 UUTuAAACGUICUAUCUCCT ST 757 AD-14172 3% 1%

BoARCULAULGEAGGIuGU Ts T TRE ACARRCCUCCARVARGUUGUTST 159 AD-14173 29% 2%

UGAGCUUARCAUAGGURART ST 760 UUuACCuAUGTuAAGCU CAT ST 76% AD-14174 659% 2%

AacucGuASiiuuiucuaAT eT 702 uARAGAGAAUUCUACGAGAUT 81 763 AD-14175 53% a% cuGoGuGchbucGGuecucTst 764 GAGGACCGACUGCACGCAGT ST 765 AD-14176 111% 4%

CACGCAGCGoCCGAGAGUAT ST 766 GACUCUCGGGCECUGCEUET ST 767 AD-14177 87% 6%

AGUACCAGGGAGACUCCGGTST 768 CCGGAGUCTUCCCUGGUARCTT ET 769 AD-14178 59% 2%

BCGGAGGAGAUAGAACGUUTST 70 ARNCGUUCUAUCUCCUCCGUT ST 771 AD-14179 o% 2%

AGARCGuULARARCGAGAUT ST TE AUCUCGUIUUARACGUUCHT ST 773 AD-14180 43% Z%

LAcGuuuAABACCAGAuCuTST 774 AGAUCUCGUUTMARACGUUT ST 775 AD-14181% 0% 10%

AGCuuGAGCUUARCAVAGG TST 778 CCuAUGUUAAGCUCRAGCUT 8T 7 AD-14182 100% 7%

AGCcuuAACAUAGGUARAVAT ST Tre VAUUUACCUAUGTuARGIUTST 778 AD-14183 60% 5%

UAGAG ul eRAARCouAUC TET 780 GRUAGGUUUUGUAGCUCUAT ST Tal AD-14184 129% 6% uAGuuGuanceccuccuuuhl eT 782 WARAGGAGGGAUACAACUAT 5T 763 AD-14185 62% 4% bochooccAGACAUCUGACUTST 784 AGUCAGAUGUCUGGEUGGUT ST 18E AD-14188 42% A%

AGAABCUARAULGALCUCET ST 186 CGAGRUCARUULAGIUUCUT8T 787 AD-14187 123% 12% uoucGuAGARLAVCUWAATST 788 JUARGALAAUUCUACGAGATST 789 AD-14188 28% 2%

CARCUUAUUGGAGGUUGUAT eT 750 LACARACCUCCARUAAGUUGT ST 791 AD-14189 13% 1%

LLGEUAUC CCUCCUUUARGUT ST 792 ACUUAAAGGAGGGALACART ST 793 AD-14100 EO% 3%

BCACRACULAUUGEAGCuUT ST 784 AACCOCCARURAGUUGUGAT ST 788 AD-14181 a3% 3%

AGAACuGUACUCURCUCAGTST THE CUSAGAAGAGuACAGITICUTST 787 AD-14192 40% B%

GAGouUAACRLAGGUARAYT ST 798 ATUUACCUAUGUUAAGCECTST 788 AD-14183 E7% 3% chgchhchucubuocuuasTst BOD CubAGGACAGAUGUUGGLGT 8) 301 AD-14194 51% 4%

ARAGCoCACURUAGAGuALTST 802 AuRCUCUARASUGGGCUUTTET 803 AD-14195 TIE 5%

AAGCoCACUUAGAGUAUAT ST 804 URAGACUCUARRGUGGGCULT ST HGS AD-14196 42% 6%

GAhccuuhuuuGGuihucuGTsT 806 oAGAUUACCARAUAAGGUCT 8T 807 AD-14197 15% 2%

GRuLRAuGuUACuCcAAGACUT aT Bog AGUCUUGAGUACATURRUCT ST 808 AD-14198 12% 2% cuLUAAGAGGCCuARAcucATsT B10 UGAGUuAGGCCUCTLAARGT 8T B21 AD-14199 18% 2% uuAfAcchAAccouhunGATaT 812 UcBANAGGGUUUCGUTUAAT 5T B13 AD-14200 72% a% weuGuuGGAGRucuAhuAAL TT 814 AULALAGAUCUCCAACAGAT AT B15 AD-14201 D% 3% cuGAuGuuucuGAGAGACuTST Ble AGUCUCUCAGRARCATCAGTST B17 AD-14202 250% 3%

GoAuvhcucuAiucGuueccTsT 818 GGEAACGACUAGAGUAUGCT ST g1a AD-14203 21% 1%

GuUCCULAUCGAGRAYCUATST 520 VAGAUUCUCGAUARBGEAAL TST 821 AD-14204 4% 2%

GohcuudGhucucuchchulsT 822 AUGUGAGAGARUCCARGUSCT ST 823 AD-14205 5% 1%

AAPARAGGRACURGRUGGCT ET 824 GCoAUCUAGUUCCUUTDUUT ST 825 AD 14206 TO% a%

single SDs dose 2nd i can SEQ Antisense sequence (3° - SEQ duplex screen § Soon

Sense sequence {5-37} ID 504 In >: 2% nm Td {among

NG. NO. name JIE sadry . residual :

HRNA] poseat

AGAGCAGAuvACcCcuLuGeGTsT 826 CGCAGAGGURAUCUGCUCUTST 827 AD-14207 55% 2%

AGeAGANUACCUCUGCGAGT ST B28 CUCGCAGAGOUARTICUGCUTST 820 AD-14208 100% 4% cocuGACAGAGUUCACAMATST 820 UUUGIGARCUCUGUCAGGET ST 831 AD-14200 34% 3%

GuuuAccGRAGuUGUUGuIULT ST 832 AARCARCACUUCGEUARACTST B33 AD-14210 13% 2%

WUACAGUACACARCARGEATST B34 UCCTICUUCTEGUACUGUART ST 835 AD-14211 O% 1%

AcCuGGALCGURRGAAGGCATST 836 UGCCUTCTUACGAUC CAB TT 837 AD-14242 20% 3%

GAGCAGRULACCUCUGCCGATST B36 UCGCAGAGEMARUCUGCUCE ST 83% AD-14213 48% 5%

AARRGEAGUUAGUGUACGATST B40 UCGUACACUAACTUUCTUUUT ST 841 AD-14214 28% 18%

GAC CAIUVARLUUGGCRGAT ST 842 UCUGCCARRTuARAUCGECT sT 843 AD-14215 132% 0%

GAGAGGAGuGAUAAuURAAT ST gad UUuaAUuATCACUCCUCTCT ST 84% AD-14216 3% 0% cuGGACGAUUGGCUGACRAT 8T 346 UUGUCAGCCARUCCUC AGT 8T 847 AD-14217 19% 1% cucuAfuctuuecchoucATsT 848 UGAGUGGEGAACGACUAGAGTST 849 AR-14218 67% 8%

GAUACCAUUACUBCAGUAGT ST 850 CulACUGUAGUAAUGERAUCT ST BEL AD-14219 76% 4%

LUCGICUGCGAAGAAGAART 8 T £252 JUUCUUCUTCGRAGACGART ST B53 AD-14220 33% B%

GAARAGARAGUUAGUGUACGT aT 854 COuACACURARCUTCIUUUCT ST 855 AD-14221 25% 2%

UGAUGUIVACCGRAGUGUUT ST 556 ARCACUUCGGUARRCRTCATST 857 Al-14222 T% 2% uGuuubucchAuucuGEAUT ET 858 AUCCAGARUUGGRACARR CAT ST 850 AD-14223 19% 2%

AUGARACAGUAUAC CuGCEAT ST B60 UCCoAGGUAUACUCUTTCAUT ST 861 AD-14224 13% 1%

GouAcucuGAuCAANGcALT aT B62 AUGCRUUGAUCAGAGUAGCT ST 863 AD-14225 15% 2%

GeoouuGuAGAAAGAACACTSY B64 GUGUUCUUGCuACRAGEGCTsT 865 AD-14226 11% C%

LCRUGUUCCULAVCGAGARTST B&O UUCUCGRUAAGGRACAUGAT ST 867 AD-14227 5% 1%

GARUAGGGURACAGAGUUGTsT B58 CARCUCUGUAACCCUAUTCT 5T 858 AD-14228 34% 3%

CARACUGGAUCGUARGARGT ST B10 CUUCTMACGATCCAGTUUGET ST B71 AD-14229 15% 2% cuuAuuuGSupducuGouGTsT B72 CAGCAGAUUACCAARURAGT ST 873 AD-14230 20% 1%

AGCRAUGUGGAARCCUBACTST B74 GULAGGUUUCCACAUUSCTT 87 875 AD-14231 18% 1%

BCARAAARGCAGACCChuUT AT 876 AXRUGECUCUGCUUwAUUGUT ST B77 AD-14232 21% 1%

AhcchouuAGuAGuUGuC CATs 578 UGEACACUACUAAGUGGTUT ST 879 AD-14233 106% 12%

AGUCRAGAGCCAUCUGUAGT ST 880 CubcAGAUGECUCUUGACUT ET 281 AD-14234 35% 3% cucccudGicuuccouhuuTsT Bgz ARUAGSCAAGUCUAGGGERGT 8T 883 AD-14235 48% 4%

ALAGCUABAURARACCARRTST 884 UUUGEUUTUAATGUAGCUAUT ST 285 AD-14236 23% 3% uGGCuSGuAvARUNCCACET 8 T BRE CGUGGARTUACACCAGC OAT ST B87 AD-14237 72% 9% uuAuvuuGGLRAGCuGcuGET ET 888 AcAGCAGAUVACCAARIART BT EER AD-14238 22% 7%

AAcuRGAUGGoUULCHCAG TST gag CUGAGARAGCCAUCUAGUUT ST 891 AD-14239 20% 2% uehuGtcGueGCAGCcARAT ET 282 TUUUGGCUGCGACGLCAUGAT ST 893 AD-14240 Ti% 6%

ACUGGAGGAUUGSCUGACAT ST 894 UBUcAGCcAAUCCUCCAGUT ST Bak AD-14241 14% 1% cubvhbunGehoulucutuTsT 896 ARAGARUAGUGCAADuALAGT aT 807 AD.14242 11% 2%

ARRGGUCACCUAALGAAGATST 898 QCUUCATWAGGUEACCUUUTST gag AD-14243 11% 1%

AuGhbuGCAuAcucudGucTaT 200 GACuAGAGUAUGCAUUCAUTST S01 AD-14244 15% 2%

AAcRUAVUGARUARGCCUBT ST 202 CAGGCURAUTCAAUAUGUTT ST BU3 AD-14245 B0% 7%

AAGAAGGCAGUUGACCARACT ST aca GUUGGUCAACUGCCUUCUUTST ans AD-14246 57% 5%

GAUACURRARGAACARUCATST 406 UGATUSUUCUGUuAGUATCT ST 8G? AD-14247 0% 3%

AuACUGARARRUCABRUAGICT ST SCE GACUATUGATIUUCAGUAUTSST aco AD-14248 39% 4%

AABAAGGARCUAGAUGGCUT ST 910 AGCCAUCUAGUUCCUULYUT ST B11 AD-14249 64% 2%

GAACUAGRLGGCULUCUCAT AT 912 UGAGAARGCCAUCUAGTUCTST 913 AD-14250 18% 2%

ChiAccuRACUGAAGRACCUTST 314 AGGUCHUcACURAGEUUUCTST 8lE AD-14251 56% 6% pAccchucARoAcuGGulA TT 916 UuACCAGUGUUGRUGGCUART ST 917 Al-14252 48% 6%

AuuuuGAuAucuAccehuuTsT 21g BAUGGGUAGAUACARMATUT ST 910 AD-14253 30% 5%

AuctouiuAGuuchounuGTeT G20 CARAGUGARCUAUAGGGAUTST 921 AD-14254 44% 2%

AuGGGCUABARUUGCACATST 92Z wAGUGCARATUAUAGCCCRUTST ‘923 AD-14255 108% a%

AGRuACcucuGeBRAGoCoTeT G24 GEGCUCGCAGRGGLUAALCTTST 955 AD-14256 1.08% 6% uAAuucCcACGuACoCuUuCATST 826 UGAAGEGUACGUGGAATUATST 527 AD-14257 23% 2%

GuctuuccchoucAGuuuuTst 228 ABRACUGACUGIGARCGACTST 929 AD-14258 21% 3%

AnhuchhucccuGuuGhAcuTsT 230 AGUeARCRGGGAUUGATITUT ST 931 AD-14259 19% 2%

LCAUAGAGCABAGRACALATET 8932 WAUSIUCUUUGCUCUAUGAT sT 933 AD-14260 10% 1% uulCUACAGUAGCACULGGT ST 934 CoRRGUGCUACUGUAGUART 7 03% AD-14261 76% 3%

AuGuGGARAcCcUBBCUGRAATST 936 UUCcAGUuAGGUUUCCACATIET 937 AD-14262 13% 2% uGUGGARRCCURRCUGRAGT ST 938 CUUCAGUUAGEUUUCCACAT 8T 936 AD-14263 14% 2%

UCUUC CUVABALUGARAGGE TST Gai CCCUUUCAUUARGGARGATST G41 AD-14264 £5% 3% uGAAGAACCuCBARGUCARTST G4z CUGACULBAGAGGUUCTUCAT sT S943 AD-14285 13% 1%

AGAGGUCUARAGUGGRAGATST 944 DCUUCCACUTMAGACCUCUT sT 945 AD-14266 18% 3%

AuvAuculhcocAusuuuculGTsT 946 CAGAARAAUGGGUAGALAUT ST 947 AD-14267 30% G%

LAAGCoUGARGUGAAUCAGT 8T 948 CUGAUUCACTUCREECULAT ST 94% AD-14268 13% . 3% }

AGAUGCAGAccAuuubAuLTET 50 ARUuARAUGGUCUGCADCUT ST 851 AD-14269 19% 4%

AGuGuuGuuuGucchALRcTsT 952 GAAUUGGACARACAACACUT ST G53 AD-14270 11% 2% culuAAuGAAGAGCruULUTsT 954 ARARRRGCUCUUCAUMAUACT 5T 355 Al-14271 11% 1%

AGAGGAGUGRLAATOABRGTST 256 CUTLwBAUVANCACUCCUCUT ST 857 AD-14272 7% 1% nuuGucuGUIACRRACEL TST 258 AUGUATUGUARCAGAGRART ST 950 AD-14273 14% 2%

ARCAUCUALARUMGCARCATST 560 UGUUGARTUAUAGAUGUUTST 961 AD-14274 73% i%

single 50s - ng

SEG 6. SED dose sereen

Sense sequence {5-3 iD prisense seguence | 1D duplex screen # (among

NC. NO. name 25 nH [% guadru residual Po

FRNA] plivat es} uGCUAGAAGUACAUAAGACT ST B62 GUCURAUGUACTUCUAGCAT ST 963 AD-14275 10% 1%

AhuGuicucAGACuGALCTST 964 GAUCAGUCUUGAGUACATUT ST 965 AD-14275 89% 2%

GuAcucAAGAcuGhAucuucTsT 968 GAAGAUcAGUCUUGAGUACT ST 967 AD-14277 TE 1%

CACUCUGAURAACUCARUGTST 968 CAUTUGAGUUUAT CAGRGTUET ST G68 AD-14278 12% 1%

ARGAGCAGAUUACCuCuGCeTST 970 SCAGACGGUARUCUGCUCTUT ST a71 AD-14279 104% 3% ueuGCcBAGCecAGAUCAACT ST 872 SUUGAUCUGGECULGCASATST 973 AD-14280 25% 2%

BAcIGAScouuGuSuAGAT ST 874 VANACACAAGGCUCARGUUT ST 475 AD-14281 43% 3%

GAAUALALAUAUCAGC CGT ST 574 CCRGCUGAUALAURLATUCT aT 277 AD-14282 45% a% uGuchuccouAuAGUUCAS TET 378 GUSAACUALAGGGAUGACATST 973 AD-14283 30% 5%

GAucuGGcAbocAuAuuneTsT 380 GARRGAUGGUUGCCAGAUCT ST a8: AD-14284 58% 3%

LEGeAAccAuARUNCUGGAT 8T 282 TCCAGRALLAUGGIRIGC CAT sT 9e3 AD-14285 48% 3%

GAuGuUUACCGAAGUGUNGT aT 884 CAACRCUHICGGUAARCAUCT ST 985 AD-14286 49% 3% vue CuAucGAGARRCUAAT ST 086 DuAGATUICUCCGAMARGGRAT ST 287 AD-14287 5% 1%

AGCUUAAULGCULUUCUGGAT ET 288 UCCAGARAGCRAULRAGCUTST 484 AL-14288 50% 2% uiGenanuAuGGEAGACCAT ST 480 UGGUCUCCoAvARGRGCBAT ST 961 AD-14289 48% 1%

SucAuGEeGucCcAGecARTET 982 FIGGCUGCGACGC CAUGACTST 203 AD-14290 112% 7% uARuuGcAcuArcuuuseGTsT 994 CGeARAGANAGUGCARADUATST ag9k AD-14291 Th 2% cuhucuunGefuanttooAt eT 946 UGGCCALACGCARAGAGAGT ST 987 AD-14292 80% 6% ueceuduAGuuchcuuuGuTsT 998 ACARAGUGAACURUAGEGATST gag AD-14293 58% 2%

VCARCCUUUBAVICACUUGT ST 1600 CARGUGAAUUARRGGUUGAT ST 1003 AD-14294 77% 2%

GGoARLCRURALUUCUGEAATST 1002 UUCCAGRAARRUATGEIUGCCT ST 1003 AD-14295 62% 2%

AGUA CUCARGRCLUGAL CUT ST 1004 ACGAUCAGYCUUGAGUACATTST 1003 AD-14296 59% 4%

GoAGAcoAnuuAALLLGEGEe TST 1008 GCCARATUARRUGGUCUGCT ST 1067 AD-14207 37% 1k ouGAGAGATUACAGALGUTST 1008 ACAUCUGUAGUCUCUCAGAT ST 1009 AD-14298 21% 1% uGCUCAUAGAGCARBGARST ST 1010 GUUCTUUGCUCUAUGAGCAT ST 101% AD-14209 6% 1%

AcAUBAGACcuuAuuuGGuTsT 1012 ACcAARURAGEUCUUALGUT ET 10.3 AD-14300 17% 2% vunGuGouGAuucuGhesGTaT 1014 CoAUCAGARUCAGCACARRT ST 1055 AD-14301 87% 6% ccluchAchCuGGUARGRATST 1Ci6 UUCULACCAGUGTUGAUGGET ST 1017 AlD-14302 13% 1%

AGACARAUUCCGGAUGUEGAT SY 1018 UCCACAUCCEGAAUUGUCET AT 1018 AD-14303 13% 3%

GAACLUGAGccuuGuUGUAUTET i0z0 AUACACBAGGCUCARGUUCTST 1022 AD-14304 30% 2%

VARULUGG CAGAGCGGARMAT 8T 1022 UUUCCGCUCUGE cARRTUAT ST 1023 AD-14305 14% 2%

LEGAUCAAGUUAVLAUGEGT 8 T 1024 CCCRubRuAACUTCAUCCATST 1025 AD-14306 22% 4%

AUCUACAUGARCUACAAGAT ST 1026 UCUUGuUAGUUCATGUAGAUTST 1027 AD-14307 28% 6%

GGuAuumIuGAUCUGG CAAT 8T 1028 TUGCCASAUCAABABLACCTST 1029 AD-14308 €2% 8% cuARNGAASAGUANACCUGT 8T 1030 CAGGUAGACUCTUcAUUAGT ST 1031 AD-14308 52% 5% wEGAGARACULVACUGAVAT ST 1032 GAUCACUBACUUUCT CARAT ST 10233 AD-14310 32% 1%

CGAUARGRUAGAAGAUCAAT ST 1034 UUGAUCUUCLAUCTUATCGT 8T 1035 AD-14311 23% 2% cuGGeAACCcALAULUCUEET ST 1636 CCAGAARUVARIGGUUGTCAGT ST iG37 AD-14312 20% 6%

GAGAUAcchuuAcuACAGUT ST 1038 ACUGUAGURAUGGUAUCUATST 102% AD-14313 co% 4%

GuiuudlAuuGtBuuuciuTsT 1040 AUGARACC CARUTIAAURACTST 1041 AD-14314 52% 3%

AAGACounluUUGGUAARCTST 1042 GAUUACCARRUAAGGUCTEUE ST 1043 AD-14315 66% 4%

GouBuuGAnAAGAGAGCue TST 1044 GAGCUCUCUuAUCAACAGCT ST 1045 AD-14316 19% 4% vAguchuGuuucucAGAuuT aT 1046 ARUCUGAGAARCAUGAGUAT ST 1047 AD-14317 16% 5%

CASAUGCACGUAACGCASCTST 1048 GUUGCCURACGUC CAUCE aT 1049 AD-14318 52% 11% uAucochACAGGUACGACATST 10380 UGUCGuACCUGUUGGGAUAT 8T 1051 AD-14319 28% 11% chuuGCuAVIAUGECATAC TST 1052 GUCUCCCAUABUAGCARIGT 3T 1053 AD-14320 52% 10% cocucAGuAbhuccAuttuTsT 1054 ACCAUGGAUUNACUGAGGET sT 1C55 AD-14321 53% 0%

GGuchAuuhcuGCacuuGuATaT 1056 UACAAGGGCAGUARTGACCT ST 1057 AD-14322 20% 2%

ABRCCACUCARAARCAMURGTST 1058 CABRAUGUUUUUGRGUGEUUTET 105% AD-14323 116% 6%

UUUGCARGUURAUGAR CUTEST 1060 AGRUTCATCRRACTHIG CARAT BT 1061 Al-14324 14% 2%

WUANLILCAGUAGUCAGART ST 106% UFC UGACUACTGRRRRUARTST 1063 AD-14325 50% 2% vutucuoGAnuchhAucuaTsT 1564 ARGAUTaGRAUCGAGARRATST 1065 AD-14326 47% 3%

GCUACGARARBRAGARGUUAGUET aT 1566 CACUAARCUUCUUUIUCGRACT ST 1467 AD-14327 18% 2%

LuuABRAACGASAucuuGeuTaT i068 AGCAAGAUCUCOTUULARAT ST 1069 AD-14328 19% 1%

GRapuGAuuAAuGuicuchTsT” 1076 UGAGUACATUMAUCAAIGCY 87 1071 AD-14328 94% 10%

GAUGGACGUAALBGCAGCuC TST 1072 GAGCUGCCUuACGUC CRUSTY 1073 AD-14330 60% 4% chucuGAcuBAuGGoucuGT aT 1074 CAGAGC CAULAGUCAGRUGT ST 1875 AD-14331 54% 7%

GuUEALCCUGUACGARAAAGAT ST 1076 UCUUGUCGUACAGGAUCAC TST 1077 AD-14332 22% 4%

AGcucuuAUnAAGGAGURAUTST 1078 AUACUCCUuAALRRGAGCUY ST i079 AD-14333 10% 10%

GoucuuAnubAGEGAGEAUATST 1080 uABACUCCUuABUARACAGCT ST 1081 AD-14334 18% 3%

LCUNAVLAAGGAGUALACGT ST 1062 CGRAUACUCCUURAAUAAGATST 1063 AD-14335 38% 6% vALUAAGGRGUAUACCGAGT ST 1684 CUCCOuAUACUCCULARVAT ST 108% AD-14336 16% 3%

CUGCRGCCCGUGAGARARAT 5T 1086 VITUUUCUcACGGGCUGCART ST 1087 AD-14337 65% 4%

VCAAGACUGAUCUUCUAAGT ET 1088 CUBAGAAGAUCASUCUUGAT eT 1082 AD-14338 18% 0%

CULCUARGUNCRCUGGARAT ST 1080 UUUCCAGUGARITUAGAAG TT 1091 AD-14339 20% 4% uGcARGuUAALGAAUCUTUUTST 1062 AARGAUUCAUUARCUUGCATST 2083 AD-14340 24% 1%

AALVCUARGGAGAUASUCARAT ST 1094 UUGACOALAUCCUDAGAUUT ST i025 AD-14341 27% 3%

AucucuGAACACAAGAACATST 1096 UEUUCTUGTGUUCAGAGAUT ST 1087 AD-14342 13% 1%

single She ar o dose nd

Sense sequence (57-3) see Antisense sequence (5 =~ a duplex screen @ among

NQ. NO. DAME 25 ot t% quadru residual as

MRNA] plicat es)

NUCUGARCAGUGEGUALCUTST 1098 AGRURCCCACUCUUCAGART 8 T 16es AD-14343 19% i%

AGUUAVULAUACCCAUCRAATST 13100 UUGAUGGGUAUARBUARCUT aT 1101 AD-14344 23% 2%

AuGouARRCUGUNCAGARATST 1102 UUUCUGRACAGUTAGLAUT ST 1103 AD-14345 20% 4%

CUBACAGAGCACUUGEUUACT ST 1104 GURARCCAAGUGCUCUGUAGT ST 1105 AD-14346 18% 2%

AUAUAUCAGCCGEGLGCGTET i108 COCGLCCEGCIGAUAIAVAT ST 1107 AD-14347 67% 2%

AuGUAARRUACGUATDUCUATST 1108 NAGAAAUACGuATTMACATT ST 1109 AD-143458 39% 3% uuu cusGhuncAiduculsT 11:0 AGAUTMGAAUCGAGAARART ST 1:11 AD-14349 B3% 6%

ARucunAAcccuuAGGACUTST 1112 AGUCCUAAGGCUGARAGATLUTST 1113 AD-14350 54% 2% counAGGAcucuSGuAUUUTaT 1114 ARBUACCAGAGTCCRARGETST 111% AD-14351 57% 8%

AAuARACUGCCoucAGuARTST ille UuACUGAGCCCAGTUTUAUU TST 1117 AD-14352 82% 3%

GAuccuGuACGARBAGEAGT ST i118 CUSCUUUUCGRACAGGAULT ST 1119 AD-14353 2% 1%

AAGRCGAURCCUGUACGAAATST 1120 UUUCGuACAGGALI CASAL ST 1121 AD-14354 10% 11%

GuGRAARCAUGECoGuueTaT 1122 GRACGGCCcRAUGUUUUCACTET 1123 AD-14355 2% 1%

CUVGAGGARACUCUGAGUATST 1124 VACUCAGAGUUUCCU cAACT SY 1125 AD-14358 0% 2% cGuuuARRACGAGAUCUNGT ST 112¢ CAAGRICUCGUUGuARAACET ST 1127 AD-14357 6% 3%

VUABAACGAGAUCUUGCUGTST 1128 CAGCRARGAUCUCSUUIVART ST 1129 AD-14358 90% ig

ARAGRUGUAUCUGGUCune TT 1130 GEAGRCCAGRUACAUCT TUT AT 1131 AD-14359 10% 1% cAGARABUGUSUCUACUGAT 8 T 1132 UGAGUAGARCACRUIUDUCUGT sT 1133 AD-14360 6% 4%

CAGGAARUGALVARUGUACT ST 13134 GuAcAUUBAUCAAUUCCTGET sT 1135 AD-14361 30% 0%

AGucbAculAAAGchubuuuTsT 1136 AALGARUGCUUGAGUHUGACUT 8T 1137 AD-14362 28% 2%

LGUGUARACRANCUACAUGAT 3 T 1138 UcAUGuAGAUTSTUACACAT 57 1138 AD-14363 50% 6%

AuAccAuwusuuccunstulsT 1140 ACCARGGAACEARUGGUAUTST 1141 AD-14364 12% 0%

GCoACADALCURAAGGEUAUARATST 1142 CAUAUCCURAGATTUCUGCT aT 1143 AD-14365 5% 2% uGGeuucucAcAGGAACucTsT 1144 GAGUUCCUGUGAGAAGC CAT ST 1145 AD- 14366 28% 5%

GAGRUGUGARRCUCUGARCTST 1148 GUUCAGAGAUUCACAUCUCT ST 1147 AD-14367 42% 4%

LGUARGC CARL GUUGUGAGT AT 1148 CUcACAECAUUSCCTuRcA TST 1149 AD-14368 93% 12%

AGCccABUGUUGUGAGGoULTaT 11350 AAGCCUCACARACATUGGCUT ST 1151 AD-14369 65% 4% uuCUGACGCu CARGUU CATT 13182 UGAACUUGARGCCUCACAAT ST 1153 AD-14370 5% 2%

AGGCAGCUCARGAGRARCATET 1154 UGUUUCUCAUGAGCUGLCUTsT 1355 AD-14371 24% 5%

AUMAANGCAVAGCACRRART ST 1156 TJUUGUGC UAT CAAUUUAUT RT 1:87 AD-14372 4% 1%

AcAARRGCUAGBACULARUTST 1158 AUNARGUUCLBGALUUUGUT TY 1359 AD-14373 3% 1%

GALALCCCARCAGGUACGATST 1180 UCGuACCUGUUGGEGAUATCTST 11el AD-14374 62% 0%

AAGuuAuuuRudcecchucATsT 1i62 UGAUGGGUAUARAGAACUTT ST i163 AD-14375 76% 4%

WEUARANACGUAMULCUAGT ST 1164 CrAGRARUACGUATUGACATST 1165 AD-14376 70% 5% weuAGuuuacAnAubAAGUTST 1166 ACTUCLAVAUGAAARCUAGAT ST 1.67 AD-14377 48% 4%

AuARAGuUAGCULCULUUALAT ST 1164 RAVARLAGAACUACTUUAUT ST lige AD-14378 48% 3% cCehAuLuGuUAGAGCUACARAT ST 1178 UFUSAGCUCUACAMAIGET 87 itl AD-14379 44% 5%

LAN CAGRAGRCAGARLT ET 1172 AUUCTGACUACUGRAARURAT ST 1173 AD-14380 35% 16%

ArhucubRAccouhfuuGuAlsT 1174 UACARCUAGGEURAGADYUTTST 117% AD-14381 44% 5%

CUUUAGAGUABACALUSGCUTST 1176 AGCARUGLAGACTCUARRGT ST 1177 AD-14382 28% 1%

AuncuGAcuARuGtoucubulsT 1178 ACAGAGCCAUUAGUCAGRUTST 1178 AD-14383 55% 13%

CACABUGAUULARGGACUGT ST 1180 CAGUCCUUABAGCAUDGUGTT 1iel AD-14384 48% 0% ucuuLUICUCGAUUCARAUT ST 1182 AUUuGARUCGAGARRARGAT ST 1183 Al-14385 36% 2% cuuuULuCuCGANUCARALUCTST 1184 GAUUUGAATCCAGARRAAGT 8T 1185 AD-14388 41% 7%

AuuuucuGouchecGAUGAGTST 1186 CUCAUCCUGAGCRAGARAADT AT 1187 AD-14387 38% 3% nuueuGeucheGAUGAGUUTST 1188 ARCUCAUCGUGRGCAGRART ST 1189 AD-14388 50% 4%

AGAGCUACRARACcubuce TST 1190 GGAUAGGTUTUSUAGCUCITST 1191 AD-14389 98% 6%

GAGCCRARGGUACACCACUTST 1192 AGUGGUGuACCUUTGGCUC TST 1183 AD-14380 43% 8%

GC CARAGGUACACCACUACTST 1194 GCuAGUGEUGUACCUTUGECT ST 1185 AD-14381 48% 4%

GAACUGUACUCUUCUCASCTST 1196 GCUSAGRAGAGUACAGUUCT ET 1187 AD~14382 44% 3%

AGGUAARUAUCACCRACAUT ST 1198 AUGUUGGUSALATUUACCUT ST 1189 AD-14303 37% 2%

AGeuAchAiAccuducouulsT 1200 AAGGAUAGGUUUUGUAGCUTST 1201 AD-14394 134% 1% uGUGARAGCRAUUNARILCCT ST 1202 GGAAULAARUGCUTUCACAT ST 1203 AD-14395 55% 4%

GoocAcuUUAGASURUACAT ST 1204 UGuAUACUCUAAAGUSGGGCTST 1205 AD-14 396 49% 5% uGuGcehchcucohAGACeTsT 1208 GGUCTUGGAGUGUGGCACATET 1207 AD-14397 1% 6%

ABRACUAARIUGALCUCGUATET 1208 GACGAGAUCARTUUAGULUT YT 1208 AD-14398 81% 7% uGAucucGurGRAuubucu Ts 1218 AGAUAATUCLACGAGAUCATST 1211 AD-14359 38% 4%

GeGUGCAGUCGGuCCuCcATsY 1212 UGGAGGACCGACUGCRCGCTST 1213 AD-14400 16% 8%

ARAGUUUAGAGACAGCUGATST 1214 UCAGAUGUCUCUARRCUTUTST 1215 AD-14401 47% 3%

CRGAAGGRRABNGUACKARTST 1216 UGUGUACARAATUCCUUCUST ST i217 AD-14402 31% 1% cGCCCCRGAGUACCAGGGAT ST 1218 UCCCUGGUACUCUCEGGCETsT 1219 AD-14403 105% 4%

CGGAGGAGAVAGEEGuuLTaT 1220 ARACGUUCuAUCUCCUCCETST 1221 AD-14404 3% 1%

AGAUACARACGUUULAARACGT ST 1222 CGUUUUAAACGUUCUAGCUT eT 1223 AD-14405 15% 1%

GGAACAGGARCRUCACAACTST 1224 GUUGUERAGTUCCUEUCCT eT 1225 AD-14406 44% o%

GCuSAGCCAAAGGUACACGATST 122% UGGEUGuACCUTUGGLCUCACTEST 221 AD-14407 41% 4%

AuccuccoubGhcuucceulsT 1228 AGGCAARSUCUAGGGAGEATTST 1229 AD-14408 104% 3%

CACACuLCRAGACCUGUES TST 1230 GCACAGGUCUUGGAGUGUSTsT 123% AD-14409 e7% 4%

ACAGARAGGARAUAUGUACAAT ST 1232 UUGuACcAGATUCCUUCUGUT ST i233 AD-14410 22% TE mrss in sense sequence (57-3) Sea Antisense sequence [(5'~ In dup ex screen 6 among ®O. NO. resigua; Guadru

MRNA] pice

UuAGAGACRucuGACLUUGT ST 1234 CAARGUCAGAUGUCUCUAAT ST 1235 AD-14411 20% 3%

DApuGAucucGuAGCAAUVATST 1236 VAALTUCUACGAGAU CAAT 8T 1237 AD-144712 31% 4% dsRNA targeting the VEGF gene

Four hundred target sequences were identified within exons 1-5 of the VEGF-A121 mRNA sequence. reference transcript is : NM._003376. 1 augaacutuc Ugcugucung Jougcauugyg agocuugccu ugcugcucta couccaccal 61 geccaaguggu CcoCaggougn acccaugdcea daaggagday ggcagaauca ucacgaagug 121 gugaaguuca uggaugucua ucagcgcagc UACUgCCAUC caaucdagac coudguggac 181 aucuuccagy aguacccuga ugagaucgag uvacaucuuca agocalccuyg ugugeccoug 241 augcgaudcd goggcugcuyg caaugacdgayg ggcecuggadu gugugeccac ugadgagucce 301 aacaucacca pgcaganlau goggarcaaz ccucaccaay gocagracaul aggagagaug 361 ageounccuac agcacaacaa augugaaugc agaccaaaga aagauagagc aagacaagaa 421 aaaugugaca agcogaggog guga (SEQ ID WO:1339)

Table 4a includes the identified target sequences. Corresponding siRNAs targeting these sequences were subjected to a bioinformatics screen.

To ensure that the sequences were specific to VEGF sequence and not to sequences from any other genes, the target sequences were checked against the sequences in Genbank using the BLAST search engine provided by NCBI. The use of the BLAST algorithm is described in Altschul et al., J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth.

Enzymal. 266:460, 1996. siRNAs were also prioritized for their ability to cross react with monkey, rat and human VEGF sequences,

Of these 400 potential target sequences 80 were selected for analysis by experimental screening in order to identify a small number of lead candidates. A total of 114 siRNA molecules were designed for these 80 target sequences 114 (Table 4b).

Table 4a. Target sequences in VEGF-121

No: in VEGF- VEGF121 mRNA Lo in VEGE- VEGF121 mRNA * 1121 ORF 5' to 3 121 QRF 5' to 3'

TID | 1 | AUGAACUWCGCUGICUOGECD [Lead | 65 | COUCTACCUCCACCAVGOCARGD) 5541 | 7 | CGRACUUUCUGCUGUCUUGEEHG [1585 | 46 | COCURCEUCCACCAUGCERRGIS] 1547 5543 | 4 | AACUDUOUGCUGUCUUGGGUGEA [1567 | 4B | CUMCCUCCACCAUGCCARGOGGD) 1524 | 5 | ACUUCUGCUGUCUUGEGUGCAD [1565 | 45 | UACCUCCACCAUGCCAAGDEGC 1545 | 6 | CUGUCUSCUGUCUUGEGNGCAY [585 | 50 | ACCUCCACCAUGCCAAGUGGUCC

T5% | s | ucuccusucuvGeUGcAUUGeR [1562 | 53 | DCCACCAUGOCRAGUGGUCCCAG 55 550 551 1552 553 1554

T555 | 16 | UCUUGGOUSCAVUGGAGECHOGE [1595 | £0 | GCCAAGUGGUCCCAGGCUGEAC 556

I557 | 18 | UOGGGUSCATUGGAGCCUUGCCD 160i | §2 | CCARGUGGUCCCAGGCUSCACT 556 1559 1562 1563 | 24 | GeaunceacccuuGecunGeuas [1607 | 68 | GEUCCCAGGCUGCACCCAVGECA

Toes | 75 | CAUUGGAGCCUUGCCUUSCUGCY [EOE | 83 | GUCCCAGACURACCAUGatAS 1565

Toe

Toe 1570 5573

T575 | 36 | UGCCUDGUGCUCUACCCCACE [L615 | 80 | CACCCAUGGCAGAAGEAGGRGEE 57e 57 1578 1575 | 40 | UUGCUGCUCUACCUCCACCAUGE [1623 | 84 | CAUGGCAGAMGGRGGAEACHGA 1580 1561 | 42 | GCUGCUCUACCUCCACCAUGCCA [1625 | 86 | UGGCAGARGGAGGAGOGCAGAR

Tea | 43 | CUGCUCUACCUCCACCAUGCCAA [1626 | 67 | GOCAGRAGGAGGRGAGCAGRAUC

EE

HO: in VEGE- VEGF121 mRNA NO: in VEGE- VEGF121 mRNA 1121 ORF 5 fo 3 121 ORF 5" to 37

Te | 6h | CAGE eeCianITT [67 | 13 | UGUCURNCAGCa CUACUREC

Te25 | 50 | AAAGeAGeAGGCAGAACATC [1675 | To6 | GOCUAUCKCACAGCUACUGECA 1630 | 51 | criceAceacoccamanCanCa [L676 | 157 | DCUABCAGCGOAGCUACUGCaAT 1631 | 57 | nsciGonocaCAGMAUCANCAC [1277 | 138 | CORUCAGCRCAGCIACURCCALC 632 ii 7635 | 56 |neeheaccatauchUCiceias [1681 | 142 | CAGCGCAGCUACUSCCAUCERAT ee

Te37 | 55 [ChocecAGATCAUCAGRGUG [1663 | 141 | GOGCAGTOACUGCCAUCCARUES 7635 | 55 | 3accaGRAIcAUCACoRaGUGG [L684 | 245 | CACAGCUMCUGCCALCERATCEA

Teas ean

Tea

Tez eos

Tes

TE

Tess

Tee:

Ted

Teed ree ee oe

Tees

Tee?

Tere

Tent er

NO: in VEGF- VEGF121 mRNA a- in VEGF- VEGF121 mRNA 121 ORF 57 fo 3' 121 ORF 5 to 3f

EE

Im

EE

ED

1728 730

Ta re

TE

173

EE

Trae

Ir re

Tra a

Tres

Tae

Trap 750

Tre

Tse 77

Tre

Tiss

Tet

S76 | 222 | eocroacuaauenccounuae [1607 | 267 | GAGGGCCUGGAGUSIGIEECC. rer res rer

Tes

HO - in VEGF- VEGF121l mRNA _ in VEGE~ VEGF121 mRNA ! 121 ORF 3 to 3' 121 CRF 3’ to 37

GE

513

Te1s

Tos 616 515

IE

IE

1676 627 1628

OE

630

TE

TE

7553 53d

EE

550

Toe 542 643

Toes

T5es

Tots

Tost

Tas

OT

550

Tos a5

Tosa

TE ase res?

WO: in VEGF- VEGF121 mRNA oO in VEGF- VEGFL21 mRNA 121 ORF 5" to 37 i121 ORF 3" fo 3 130% | 36s |ucoucnecAcechmmbGoeR

To05 | 366 | coonascncancaanueuea

Ts07 | se |oncnachcaacamGumaee 1505 | 570 | caccacmcamaeoemabecas 1510 | 571 | accrcancamavaveannscah |_| |]

Toni | 57 |ecacmcmmvavamtecacac | ||] 152 | 375 |cacmacmavesmavecacace | |] 1914 | 575 | cancamavauennvacagoce |_

Tous | sve | macmmevmamacseocan |||]

Tic | 377 | acmsveucmvecAGacamaG

Lois | 37s | amveveasvecaGaceRRGA | |]

T533 | 364 | vemawecheacCmacmaana | | |] 1505 | 385 |moccaccommmieroage

Tone | 367 [AvecsaccamGamnGGe yo27 | 388 | vecaenccammsmmaGamAGRe | ||] 1928 | 385 | coaGacCammsamAGRUAGRGCA

Ts29 | 390 | Caorccmmaammaentmacean | | |] 130 | 391 |ataccamemiemeacme

Tosi | 307 |aacoumemcanamanomaca 1932 | 393 | mccamacmneavamecmRGac 1553 | 394 | commeamacavrescaGaca

Toss | 59s | crmsemmcavaGaccaGacan 1535 | 396 | mmmemmnauacaccmgacaas | ||] 1536 | 351 | amcamncauacaccangaCaaGE] |_ 1557 | 398 | memmacamameceeataaein | |__

To3s | 39% | camseavncaccaaaaciia 1535 | avo | mancavaGaecaRGACRRGRRRA

Table 4b: VEGF targeted duplexes

Strand: S= sense, AS=Antisense

BE [een FE] mow onin| ID Duplex ID d > Strand Sequences - JORF| NO: (5-3) 0: 1 [2184 AUGRACUTUCUGCEGUCUUGEED AL-DP-4043 | 8 [1940] 5 GAACUDUCUGCUGUCUUGGEGU 3

AS [1%41]3 UACUUGARAGACGACAGARACCCA 5

D2 12185 GUGCAUUGEACCCUUGCCUUGCY AL-DP-4077 | 8 }1942| 5 GCAUUGGAGCCUUGCCUUGCT 3

AS [1943|3 CACGUARCCUCGGRACGGAACSA 5 47 [2186 [UCUACCUCCACCAUGCCAAGUGG [AL-DE-4021 | 5 {1%24] 5 UACCUCCACCAUGCCAAGUTT 3 2S [19453 TTAUGGAGGUGGUACGGUUCA 5 48 [2187 [CUACCUCCACCAUGCCARGUGED [AL-DP-4109 | 5 1946] 5 ACCUCCACCAUGCCAAGUGTT 3 25 |1947{3 TTUGGAGGUGGUACGGUUCAC 5 50 [2188 IACCUCCACCAUGCUAAGUGGUCC JAL-DP-4006 | S [1948] 5 CUCCACCAUGCCAAGUGGUCC 3

AS [1949|3 UGGAGGUGRUACGGUUCACCAGS 5

AL-DP-40B83 | 5 [1930] 5 CUCCACCAUGCCAAGUGGUTT 3 25 11951]3 TTGAGGUGGUACGGUUCACCA 5 51 [2189CCUCCACCAUGCCAAGUGGUCCC AL-DP-4047 | 5 [1952] 5 UCCACCAUGLCAAGUGGUCCC 3

AS [1953|3 GGAGGUGGUACGGUUCACCAGGE 5

AL-DP-4017 | § [1954] 5 UCCACCAUGCCAAGUGGUCTT 3

AS 19553 TTAGGUGGUACGGUUCACCAG 5 52 [2190 jCUCCACCAUGCCAAGUGSUCCCA -DP-4048 | 5 1855) 5 CCACCAUGCCAAGUGGUCCCA 3

AS [19573 GAGGUGGUACGGUUCACCAGGGY 5 nL-DP-4163 | 5 {1958| 5 CCACCAUGCCAAGUGGUCCTT 3 25 [18593 TTGGUGGUACGGUUCACCAGE 5 53 12192 [UCCACCAUGCCAAGUGGUCCCAG RL-DP-4035 | 8 1960! 5 CACCAUGCCAAGUGGUCCCAG 3

AS 11961 |3 AGGUGGUACGGEUUCACCAGGGUC 5

AL-DP-4018 | § [1962] 5 CACCAUGCCAAGUGGICCCTT 3

AS 196313 TTGUGGUACGGUUCACCAGGS 5 54 [2192 ICCACCAUGCCARGUGGUCCCAGG RL-DP-4036 | § [1964] 5 ACCAUGCCAAGUGGUCCCAGSE 3 2s 19653 GGUGGUACGGUUCACCAGGGUCE 5 -DP-2084 § 11966] 5 ACCAUGCCAAGUGGUCCCATT 3

AS [19673 TIUGGUACGGUUCACCAGGGU 3

BE |e [TE] mee onin{ ID Duplex ID a iD Strand Sequences

ORF| NO: (57-37 NO: 55 [2193 [CACCAUGCCAAGUGGUCCCAGGC AL~DP-40093 | 3 [1968] 5 CCAUGCCAAGUGGUCCCAGGC 3

AS 196% |3 GUGGBUACGGUUCACCAGGGUCCE 5

RL-DP-4085 | S 1370] 5 CCAUGCCARGUGGUCCCAGTT 3

AS 119713 TTCGUACGGUUCACCAGGGUC 5 56 [2194 RCCAVGCCAAGUGGUCCCAGGCY RL-DP-4037 | 8 11972] 5 CAUGCCRAGUGGUCCCAGGCU 3

AS |19273|3 UGGUACGGUUCACCRGGGUCCGA 5

LL-DP-4054 | S [1974] 5 CAUGCCRAGUGGUCCCAGGTT 3

AS [1975{3 TTGUACGGUUCACCAGGGUCC 5 57 2195 |CCAUGCCAAGUGGUCCLAGGCUG RL-DP-4038 | 5 [1976] 5 AUGCCAAGUGGUCCCAGGCUG 3

AS [19773 GGUACGGUUCACCAGGGUCCGAL 3

AL-DP-4086 | 5 1878] 5 AUGCCARAGUGGUCCCAGGCTT 3

AS [19793 TTUACGGUUCACCAGGGUCCG 3 58 [2196 CAUGCCAAGUGGUCCCAGGLUGE AL-DP-4049 | S 11980] 5 UGCCAAGUGGUCCCAGGCUGC 3

AS [19813 GUACGGUUCACCAGGGUCCGACG 3

AL-DP-4087 | S 1982 5 UGCCRAGUGGUCCCAGGCUTT 3

AS {1983|3 TTACGGUUCACCAGGGUCCGA 5 58 [2197 AUGCCAAGUGGUCCCAGGCUGCA BL-DP-4001 § S$ 11984] 5 GUCRAGUGGUCCCAGGCUGCA 3

AS [198513 UACGGUUCACCAGGGUCCGACGU 5

AL-DP-4052 | A [1986] 5 GCCAAGUGGUCCCAGGCUGTT 3

AS [19873 TICGGUUCACCAGGGUCCGAC 5 2198 [UGCCAAGUGGUCCCAGGCUGCAC BAL~DP-4007 | 8 1988] 5 CCAARGUGGUCCCAGGCUGCAC 3

AS 11583813 ACGGUUCACCAGGGUCCGACGUG 5

LL-DP-4088 | 3S |1990] 5 CCAAGUGGUCCCAGGCUGCIT 3

AZ 198113 TTGGUUCACCAGGGUCCGACE 3 61 |2199iGCCRAAGUGGUCCCAGGCUGCALC BL-DP-4070 | 5 |1992} 5 CRAGUGGUCCCAGGCUGCACC 3

AS [18933 CGGUUCACCAGGGUCCGACGUGE 5

RL-DP~-4055 | S [1994] 5 CAAGUGGUCCCAGGCUGCATT 3

AS {1983{3 TTGUUCACCAGGGUCCGACGU 5 l62 2200 [CCARGUGGUCCCAGGCUGCACCC |BL-DP~4071 | s 1996] 5 AAGUGGUCCCAGGCUGCACCC 3

AS [199713 GGUUCACCAGGGUCCGACGUGEE 5

GE To [ee [OE] eee on in| ID Duplex ID 4 iD Strand Sequences

ORF| NO: | (5-39 NO:

AL-DP-4056 8S 1998] 5 AAGUGGUCCCAGGCUGCACTT 3

AS {1999 l3 TTUUCACCAGGGUCCGACGUG 5 &3 2201 |CBAGUGGUCCCAGGCUGCACCCA AL-DP-4072 5 [2000 5 AGUGGUCCCAGGCUGTACCCA 3

AS [20013 GUUCACCAGGGUCCGACGUSGGGU 5

AT-DE-4057 8 [2002 5 AGUGGUCCCAGGCUGCACCTT 3

AS [2003[3 TTUCACCAGGGUCCGACGUGG 3 2202 BAGUGGUCCCAGGCUGCACCCARY AL~DP-4066 S j2004 5 GUGGUCCCAGGCUGCACCCTT 3

AS [20053 TTCACCAGGGUCCGACEUGES 5 2203 AGGGCAGRAUCAUCACGAAGUGSG AL-DP-4022 S 2006 5 GGCAGAAUCAUCACGAAGUTT 3

AS [20073 TTCCGUCUUAGUAGUGCUUCA 5 100 2204 [GGGCAGRAUCAUCACGAAGUGGU [AL~DP-4023 S$ [2008 5 GCAGRAUCAUCACGAAGUGTT 3

AS 200813 TTCGUCUUAGUAGUGCUUCAC 5 101 [2205 GGCAGRAUCAUCACGARGUGGUS AL-DE~4024 5 2010 5 CAGAAUCAUCRCGRAGUGGTT 3

AS |2011§3 TTGUCUUAGUAGUGCUUCACT 5 102 2206 |GCAGRAUCAUCACGRAGUGGUGA AL-DP-4076 8 z012 5 AGARUCAUCACGRAGUGGUGA 3 [20133 CGUCUUAGUAGUGCUUCACCACU 5

AL~DE-4019 S 2014 5 AGARUCAUCACGAAGUGGUIT 3

AS [20153 TTUCUUAGUAGUGCUUCACCA 5 103 R207 [CAGRAUCAUCACGAAGUGCUGAA AL~DP-4025 1 § [2016 5 GAAUCAUCACGAAGUGGUGTT 3

AS [20173 TTCUUAGUAGUGCUUCACCAC 5 1 04 2208 RGRAUCAUCACGAAGUGSUGAARG AL~DP-4110 | § 20 18 5 RAUCAUCACGAAGUGGUGATT 3

AS 1201513 TTUUAGUAGUGCUUCACCACT 5 105 [2209 [GAAUCAUCACGAAGUGGUGAAGU RL-DF-4068 5 2020 5 AUCAUCACGRAGUGGUGAATT 3

AS [202113 TTUAGUAGUGCUUCACCACUY 3 113 {2210 ACGARGUGGUGAAGUUCAUGGAU [AL~DP-4078 | 5 2022 5 GAAGUGGUGAAGUUCAUGGRU 3 2S {2023|3 UGCUBCACCACUUCARGUACCUAR 5 121 2211 {GUGARAGUUCAUGGAUGUCUAUCR RL-DP-4080 § 2024 5 GAAGUUCAUGGAUGUCUAUCA 3 2S |2025{3 CACUUCAARGUACCUARCAGAUAGU 5 1259 12212 CAUGGAUGUCUAUCAGCGCAGCU BL~DP-4111 | § [2026 5 UGGAUGUCUAUCRGCGCAGTT 3 25 |2027 [3 TTACCUACAGAUAGUCGCGUC 5

BE Tem | ew [TE meen on inj ID Duplex ID d 11) Strand Sequences

ORF| NO: (57-37 0: 130 2213 [RUGGAUGUCUAUCAGCGCAGCUA AL-DP-4041 | § 12028) 5 GGAUGUCUAUCAGCGCAGCUA 3

AS 1202913 UACCUARCAGAUAGUCGCGUCGAU 5

AL-DP-4062 | $5 2030] 5 Ty — 3 : AS j12031|3 TTCCUACAGAUAGUCGOGUCG 5 1.31 2214 UGGAUGUCUAUCAGCGCAGCUAC BL-DP-4069 | S [2032] 5 GAUGUCUAUCAGCGCAGCUTT 3

AS [2033|3 TTCUACAGAUAGUCGCGUCGA 5 132 2215 a —— RL-DEF-4312 | S (2034) 5 ARUGUCUAUCAGCGCAGCUATT 3

AS |2033|3 TTUACAGAUAGUCGCGUCGAU 5 133 2216 GAUGUCUAUCAGCECAGCUACUG BRL-DP-4026 | S [203¢] 5 UGUCUAUCAGCGCAGCUACTT 3

AS j2037|3 TTACAGAUAGUCGCGUCGAUG 5 134 12217 RUGUCUAUCAGCGCAGCUACUGE BL~DP-4095 | S [2038 5 GUCUAUCAGCGCAGCUACUGC 3

AS [2039213 UACAGAUAGUCGCGUCGAUGACE 5

AL~-DP-4020 S 12040 5 GUCUAUCAGCGCAGCUACUTT 3

AS |2041|3 TTICAGRUAGUCGCGUCGAUGAE 5 135 {2218 [UGUCUAUCAGCGCAGCUACUGCC AL~DP-4027 | 8 [2042] 5 UCUAUCAGCGCAGCUACUGTT 3

AS [20433 TTAGAUAGUCGCGUCGAUGAD 5 144 12219 |GCGCAGCUACUGCCAUCCARUCS AL-DP-4081 | S [20441 5 GCAGCUACUGCCAUCCARUCG 3

AS [20433 CGCGUCGAUGACGGUAGGUUAGC 5 146 2220 {GCAGCUACUGCCAUCCRAUCGAG RL-DP-4098 | S 2046) 35 AGCUACUGCCAUCCAAUCGAG 3

AS i2047|3 CGUCGAUGRCGGUAGGUUAGCUC 5 149 1221 [ACUACUGCCAUCCARUCGAGACE AL-DP-4028 | § 2048] 5 UACUGCCAUCCRAUCGAGATT 3

AS [204913 TTAUGRCGGUAGGUUAGCUCY 3 150 [2222 [CUACUGCCAUCCARUCGAGACCS AL~DP-4029 | 5 2050] 5 ACUGCCAUCCAAUCGAGACTT 3 } ] AS [205113 TTUGACGGUAGGUUAGCUCUG 5 151 2223 MACUGCCAUCCAAUCGAGACCCU AL~DP-4030 | 8 2052! 5 CUGCCAUCCARUCGAGACCTT 3

AS [20533 TTGRCGGUAGGUUAGCUCUGG 5 152 2224 [ACUGCCAUCCAAUCGAGACCCUG [AL-DP~4031 | S [2054] 5 UGCCAUCCARUCGAGACCCTT 3

AS |2055§3 TTACGGUAGGUUAGCUCUGGSE 5 166 2225 a — AL-DP-4008 | S [2056] 5 GACCCUGGUGGACAUCUUCCA 3

AS 2037 3 CUCUGGGACCRCCUGUAGRAGGY 5

ZH Ia a oninf ID Duplex 1D dq I Strand Sequences

ORF} NO: (53-39) NC:

AL-DP-4058 | 5 [2058] 5 GACCCUGGUGGACAUCUUCTT 3

AS [205%|3 TTCUGGGACCACCUGUAGAAG 5 167 [2226 AGACCCUGGUGGACAUCUUCCAG AL-DP-4009 | 8 2060] 5 ACCCUGGUGGACAUCUUCCAG 3

AS 12061|3 UCUGGGACCACCUGURGRAGGUC 5

BL-DP-40589 | 5 [2062] 5 ACCCUGGUGGACAUCUUCCIT 3 235 120633 TTUGGGACCACCUGUAGAAGG 5 168 |2227{GACCCUGGUGGACAUCUUCCAGG RL-DP-4010 | 5 20641 5 CCCUGGUGGACAUCUUCCAGG 3

AS [20653 CUGGGACCACCUGUAGAAGGUCC 5

AL~-DP-4060 { 5 [2066] 5 CCCUGGUGGACAUCUUCCATT 3

AS [20673 TTGGGACCACCUGUAGRAGSU 5 169 [2228 [ACCCUGGUGSACAUCUUCCAGGA BL-DP-4073 | § 2068] 5 CCUGGUGGACAUCUUCCAGGA 3

AS 206913 UGGGACCACCUGUAGRAGGUCCY 5

AL-DP-4104 | 8 [2070] 5 CCUGGUGGACAUCUUCCAGTT 3

AS 2071{3 TTGGACCACCUGUAGAAGGUC 5 170 2229iCCCUGGUGGACAUCUUCCAGGAG AL~-DP~4011 | 5 [2072] 5 CUGGUGGACAUCUUCCAGGAG 3

AS {2073|3 GGGACCACCUGUAGRAGGUCCUC 5

RL~-DP-4089 | S |2074F 5 CUGGUGGACATCUUCCAGETT 3 . AZ 2075(3 TTGACCACCUGUAGAAGGUCT 5 171 [2230 CCUGGUGGACAUCUUCCAGGAGY BL-DP-4074 | § [2076] 5 DGGUGGACAUCUUCCAGGAGU 3

AS [207713 GGACCACCUGUAGARAGGUCCUCA 5

LL-DP-4090 § 3 2078) 5 UGGUGGACAUCUUCCAGGATT 3

AS [2078|3 TTACCACCUGUAGAAGGUCCU § 172 2231 CUGEUGGACAUCUUCCAGGAGU AL-DP-4038 | 3 [2080] 5 GGUGGACAUCUUCCAGGAGUR 3

AS [20813 GACCACCUGUAGRAGGUCCUCAU 5

AhL-DP-4091 5 2082 5 GGUGGACAUCUUCCAGGAGTT 3

AS [2083]3 TTCCACCUGUAGSRRGGUCCUC 5 175 [2232 [GUGGACAUCUUCCAGGAGUACCC |AL~DP-4003 | 5 [2084 5 GGACAUCUUCCAGGAGUARCCC 3

A8 |2085F 3 CCUGUAGAAGGUCCUCAUGGG 5

AL-DP-4116 | § 2086] 5 GGACAUCUUCCAGGAGUACCC 3

RS |2087F 3 CCUGUAGARAGGUCCUCAUGGGE 5 g9

GE To | ew [CE] ewe on inj ID Duplex ED d LD Strand Sequences

ORF: NO: (37-37) INO:

AL-DP-4015 | 5 2088) 5 GGACAUCUUCCAGGAGUACTT 3

AS [208913 TTCCUGUAGAAGGUCCUCAUG 5

AL-~DP-4120 | 3 [2080] 5 GGACAUCUUCCAGGAGUAC 3

AS |2091] 3 CCUGUAGRAGGUCCUCAZUG 5 179 [2233 ACAUCUUCCAGGAGUACCCUGAU AL-DP-40989 | 5 2082] 5 AUCUUCCAGGAGUACCCUGRU 3 48 209313 UGUAGAAGGUCCUCAUGSEGACUA 5 191 [2234 RGUACCCUGAUGAGAUCGAGUAC AL-DP-4032 | 5 [2094] 5 UACCCUGRUGAGAUCGAGUTT 3

AS [2093]3 TTAUGGGACUACUCUAGCUCA 5 192 2235 IGUACCCUGAUGAGAUCGAGUACA RL~DP~4042 | 5 [2086] 5 ACCCUGAUGAGAUCGAGUACA 3

AS [20973 CAUGGGACUACUCUAGCUCARUGE 5

AL~-DP-4063 | 8 j2098}F 5 ACCCUGAUGAGAUCGAGUATT 3

AS 209513 TTUGGGACUACUCUAGCUCAU 5 209 [2236 JAGUACAUCUUCAAGCCAUCCUGU AL-DP-4064 | § [2100] 5 UACAUCUUCRAGCCAUCCUTT 3

AS |2101|3 TTAUGUAGAAGUUCGGUAGGA 3 260 12237 [GCARUGACGAGGECCUGGAGUGU RAL~DP-4044 | S [2102] 5 AAUGACGAGGGCCUGGAGUGU 3 2s 21031{3 CGUUACUGCUCCCGGACCUCECA 5 263 12238 RUGACGAGEGCCUGGAGUGUGUG [AL-DP~4045 | § [2104] 5 GACGAGGGCCUGGAGUGUGUG 3

AS 2105|3 UACUGCUCCCGGACCUCACACAC 5 279 12239 UGUGUGCCCACUGAGGAGUCCA AL-DP-4046 | 5 2106} 5 ep p— 3

AS 21073 CACACACGGEUGACUCCUCAGGU 3 281 12240 GUGUGCCCACUGAGGAGUCCAAC AL-DP~4096 | S [2108] 5 GUGCCCACUGAGGAGUCCARC 3

AS 1210913 CACACGGGUGACUCCUCAGGUUG 3 283 [2241 |GUGCCCACUGAGGAGUCCARCAY WL-DP~4040 | § [2116 5 GCCCACUGAGGAGUCCARACAU 3

AS [211143 CACGGGUGACUCCUCAGGUUGUA 5 289 12242 |ACUGAGGAGUCCAACAUCACCAD AL-DP-4065 | § 2112] 5 UGAGGAGUCCAACAUCACCTT 3

AS 1211313 TTACUCCUCAGGUUGUAGUGG 5 302 [2243 ACAUCACCAUGCAGAUUAUGCGS ARL-DP-4100 | § 2114} 5 AUCACCAUGCAGAUUAUGCGG 3

AS [2115{3 UGUAGUGGUACGUCUARAUACGCC 5 205 [2244 UCACCAUGCAGAUUAUGCEGAUC ML-DP-4033 | § 2116) 5 ACCAUGCAGAUUAUGCGGATT 3

AS [2117]3 TTUGGUACGUCUARUACGCCU 5

CL a i A ll onin| ID Duplex TB d 1 Strand Sequences

ORF] NO: (5-39 ING: 310 [2245 |AUGCAGAUUAUGCGGAUCAARCC [RL-DP-4101 | § [2118] 5 GCAGAUUAUGCGGAUCAARCC 3

AS [21193 UACGUCURAUACGCCUAGUUUGS 5 312 [2246 |GCAGAUUAUGCGGAUCARACCUC AL-DP-4102 | 5 2120) 5 AGAUURUGCGGAUCAARCCUC 3

AS [21213 CGUCURAUACGCCUAGUUUGGAG 5 315 [2247 |GAUUAUGCGGAUCARACCUCACC BRL~DP-4034 | $s 12122] 5 UUAUGCGGAUCARACCUCATT 3

AS {212313 TTAAUACGCCUAGUUUGGAGU 5 216 [2248 AUUAUGCGEAUCAARCCUCACCE AL~-DP-4113 | 8 [2124] 5 UVAUGCGGAUCAARRCCUCACTT 3

AS [212513 TTAUACGCCUAGUUUGGAGUG 5 317 [224% [UUAUGCGGAUCARACCUCACCAR AL-DP~4114 | § [2126] 5 AUGCGGAUCAAACCUCACCTT 3

AS |2127]3 TTUACGCCUAGUUUGGAGUGG 5 319 [2250 [AUGCGGAUCARACCUCACCAAGG RL-DP-4002 | 8 2128] 5 GCGGAUCAAACCUCACCARGG 3

AS [212813 UACGCCUAGUUUGGAGUGGUUCC 5

LL-DP-4115 | § [2130] 5 GCGGAUCAARCCUCACCAZ 3

AS 2131} 3 CGCCUAGUUUGGAGUGGUU 5

AL-DP~4014 S j2132 5 GCGGAUCAMRACCUCACCARTT 3

AS [21333 TTCGCCUAGUUUGGAGUGGUU 5

AL-DP-4119% § § [2134] 5 GCGGAUCAAACCUCACCARZ 3

AS |2135)1 3 CGCCUAGUUUGGAGUGGUY 3 221 [2251 [BCGGAUCARACCUCACCARGGCC BL-DP~4013 | S [2136] 5 GGAUCAAACCUCACCAAGGCC 3

AS [21373 CGCCUAGUUUGGAGUGEUUCCGE 5 341 [2252 |GCCAGCACAUAGGAGAGAUGAGC PEL-DP-4075 | § [2138] 5 CAGCACAUAGGAGAGAUGAGC 3

AS 2139{3 CGRUCGUGUAUCCUCUCUALUCG 5

AL-DP-4105 | 5 [2140 5 CAGCACAUAGGAGAGAUGATIT 3

AS 21413 TTGUCGUGUAUCCUCUCUACU 35 342 2253 |CCAGCACAUAGGAGAGRUGAGCY BAL-DP-4050 | 5 [21421 5 AGCACRUAGGAGAGARUGAGCU 3

AS {214313 GGUCGUGUAUCCUCUCUACUCGA 5

AL-DP~4106 | S [2144] 5 AGCACAUAGGAGAGAUGAGTT 3

AS [2145[3 TTUCGUGUAUCCUCUCUACUC 5 343 2254 [CAGCACAUAGGAGAGRAUGAGCUU RL-DP-4094 | S 2146} 5 GCACAUAGGAGAGAUGAGCUU 3

AS [21473 GUCGUGUAUCCUCUCUACUCGRA 5

BE Ta |e WE] ee onin| ID Duplex ID a Strand Sequences

ORF} NO: (5-3) ING:

AT,-DP-4118 S |2148 5 GCACAUAGGAGAGAUGAGCUU 3

AS 2148} 3 CGUGUAUCCUCUCUACUCGAR 3

AL-DE-4107 | 8 [2150} 5 GCACAUAGGAGAGAUGAGCTT 3

AS 1215113 TTCGUGUAUCCUCUCUACUCG 5

AL.-DP-4122 8 2152 5 GCACAUAGGAGAGAUGAGC 3

AS 2153{ 3 CGUGUAUCCUCUCUACUCG 5 344 [2255 AGCACAUAGGAGAGAUGAGCUUC AL-DP-4012 | S 2154] 5 CACAUAGGAGAGAUGAGCUUC 3

AS |21355|3 UCGUGUAUCCUCUCURCUCGAAG 5

AL-DP-4108 S$ [2156 5 CACAUAGGAGAGAUGAGCUTT 3 i AS 12157|3 TTGUGUAUCCUCUCUACUCGA 5 346 [2256 [CACAUAGGAGAGAUGAGCUUCCU RL-DP-4051 | § [2158] 5 CAURGGAGAGAUGAGCUUCLCU 3

AS [21553 GUGSUAUCCUCUCUACUCGAAGGA 5

AL-DP-4061 Ss 2160 5 CAURGGAGAGAUGAGCUUCTT 3

AS [216113 TTGUAUCCUCUCUACUCGZAG § 349 [2257 AUAGGAGAGAUGAGCUUCCUACA BL-DP-4082 | 5 [2162] 5 AGGAGAGAUGARGCUUCCUACA 3

AS [21633 UAUCCUCUCUACUCGAAGGAUGU 5 369 [2258 ACAGCACAACAAAUGUGAAUGCE AL-DP-4079 | 5S [2164 5 AGCACAACAAAUGUGAAUGCA 3

AS 2163513. UGUCGUGUUGUUUACACUUACGU 3 372 250 [SCACABCARAUGUGAAUGCAGAC AL-DP-40987 | S |2166| 5 ACAARCARAUGUGAAUGCAGAC 3

AS [21673 CGUGUUGUUUACACUUACGUCUG 5 379 [2260 |AAAUGUGARUGCAGACCAAAGAR RL-DP-4067 | § 2168] 5 AUGUGAAUGCAGACCARAGTT 3

AS 216903 TTUACACUDACGUCUGGUUUC 5 380 [2261 AAUGUGAAUGCAGACCAARGARA RAL-DP-4092 | 5 2170} 5 UGUGARAUGCAGACCARBRGATT 3

AS 217113 TTACACUUACGUCUGGUUUCU 5 381 [2262 IAUGUGRAUGCAGACCARAGLAAG RL-DE~4004 | § [2172] 5 GUGRAUGCAGACCAAAGARRG 3

AS [2173[3 UACACUUACGUCUGEUUUCUUUC 5

AL~DP-4117 | 8 12174] 5 GUGAAUGCAGACCRRAGARAG 3

AS [2175] 3 CACUUACGUCUGGUUUCUUUC 5

AL-DP-4016 § S [2176] 5 GUGAAUGCAGACCARAGAATT 3

AS 217713 TTCACUUACGUCUGGUUUCTU 5

GE IT eee [OE] ee enin| ID Duplex ID d iD Strand Sequences

ORF| NO: (5-37 O: t AL~-DP-4121 | 3 (2178) 5 GUGAARUGCAGACCAAAGAR 3

AS 2175) 3 CACUUACGUCUGGUUUCUU 5 383 [2263 |GUCAAUGCAGACCARAGAARGAU AL-DP-4005 | § |2180} 5 GRAUGCAGACCAARGAARAGAU 3

AS [21813 CACUUACGUCUGGUUUCUUUCUA 5 . RL-DP-4053 | 5 [2182 5 pp 3

AS [21833 TTCUUACGUCUGGUUUCUUUIC 5

Example 2. Eg5 siRNA in vitro screening via cell proliferation

As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, ef af [2002]

Biotechniques 33: 1244-8), a cell viability assay was used for siRNA activity screening.

HeLa cells (14000 per well {Screens 1 and 3] or 10000 per well [Screen2])) were seeded in 96-well plates and simultaneously transfected with Lipofectamine 2000 (Invitrogen) at a final siRNA concentration in the well of 30 nM and at final concentrations of 50 aM (1% screen) and 25 nM (2™ screen). A subset of duplexes was tested at 25 nM in a third screen (Table 5).

Seventy-two hours post-transfection, cell proliferation was assayed the addition of

WST-1 reagent (Roche) to the culture medium, and subsequent absorbance measurement at 450 nm. The absorbance value for contro! (non-transfected) cells was considered 100 percent, and absorbances for the SIRNA transfected wells were compared to the control value.

Assays were performed in sextuplicate for each of three screens. A subset of the siRNAs was further tested at a range of siRNA concentrations. Assays were performed in HeLa cells (14000 per well; method same as above, Table 5).

Table 5: Effects of Eg5 targeted duplexes on cell viability at 250M. 1 Relativeabsorbanceatd0mnm [0 | |] ["Sereent | | Sereentt | | Screenlll [

AL-DP-6226

AL-DP-6227 66 27, 9% [a | 108 | 33

AL-DP-6228 28 6 22

ALDP-6220 | 17 | 3 [031 7 9 | 48 | 13

ALDP-6230| 48 | 8 | 75 11

AL-DP-6231

AL-DP-6232 6 | 2

ALDP6233] 31 + 9 | 37 | 6 | 49 | 12

AL-DP-6234 103 | 40 14] 29

AL-DP-6235 107 34

AL-DP-6236 54 12

AL-DP-6237

ALDP-6238| 64 | 9 | 103 | 10 | 105 | 24

ALDP-6239 | 9 1 1 [20 | 4 [ 31 [11

AL-DP-6240 139 16

ALDP-6241] 43 1 9 [ sa © 12 | 66 [19

ALDP6242] 6 1 1 1 15 | 7 | 3 | 8

AL-DP-6243 7

AL-DP-6244

ALDP6245] 25 | 4 1 45 I 10 [ s8 | 9

ALDP6246] 34 [8 | 6 1 10 | 66 | 13

ALDP-6247 | 53 | 6 I 78 [ 14 | 105 | 20

ALDP-6248 | 7 | 0 | 02 7 | 39 |12

ALDP-6249] 36 | 8 | 48 | 13 [ el | 7 ;

The nine siRNA duplexes that showed the greatest growth inhibition in Table 5 were re-tested at a range of siRNA concentrations in HeLa cells. The siRNA concentrations tested were 100 nM, 33.3 aM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.046 nM.

Assays were performed in sextuplicate, and the concentration of each siRNA resulting in fifty percent inhibition of cell proliferation (ICso) was calculated, This dose-response analysis was performed between two and four times for each duplex. Mean ICsq values (nM) are given in

Table 6.

Table 6: IC50 of siRNA: cell proliferation in Hela cells 19

Example 3. Eg5 siRNA in vitro screening via mRNA inhibition

Directly before transfection, HeLa S3 (ATCC-Number: CCL-2.2, LCG Promochem

GmbH, Wesel, Germany) cells were seeded at 1.5 x 10% cells / well on 96-well plates (Greiner Bio-One GmbH, Frickenbausen, Germany) in 75 ul of growth medium (Ham’s F12, 10% fetal calf serum, 100u penicillin / 100 ug/ml streptomycin, all from Bookroom AG,

Berlin, Germany). Transfections were performed in quadruplicates. For each well 0.5 ul

Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 ul Opti-

MEM (Invitrogen) and incubated for 15 min at room temperature. For the siRNA concentration being 50 nM in the 100 ul transfection volume, 1 ul of a § uM siRNA were mixed with 11.5 pl Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. siRNA-Lipofectamine2000- complexes were applied completely (25 pl each per well) to the cells and cells were incubated for 24 h at 37°C and 5 % CO; in a humidified incubator {Heroes GmbH, Hanan).

The single dose screen was done once at 50 nM and at 25 nM, respectively.

Cells were harvested by applying 50 ul of lysis mixture (content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA} to each well containing 100 pl of growth medium and were lysed at 53°C for 30 min. Afterwards, 50 ul of the lists were incubated with probesets specific to human EgS and human GAPDH and proceeded according to the manufacturer's protocol for QuantiGene. In the end chemoluminescence was measured in a

Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RL Us (relative light units) and values obtained with the hEg5 probeset were normalized to the respective GAPDH values for each well. Values obtained with siRNAs directed against EgS were related to the value obtained with an unspecific siRNA {directed against HCV) which was set to 100% (Tables 1b, 2b and 3b).

Effective siRNAs from the screen were further characterized by dose response curves.

Transfections of dose response curves were performed at the following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77 picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9

IM and mock (no siRNA) and diluted with Opti-MEM to a final concentration of 12.5 ul according to the above protocol. Data analysis was performed by using the Microsoft Excel add-in software XL-fit 4.2 (JDBS, Guildford, Surrey, UK) and applying the dose response model number 205 (Tables 1b, 2b and 3b).

The lead siRNA AD12115 was additionally analyzed by applying the WST- proliferation assay from Roche (as previously described).

A subset of 34 duplexes from Table 2 that showed greatest activity was assayed by transfection in Hela cells at final concentrations ranging from 100nM to 10fM.

Transfections were performed in quadruplicate. Two dose-response assays were performed for each duplex. The concentration giving 20% (1C20), 50% (1C50} and 80% (1C80) reduction of KSP mRNA was calculated for each dupiex (Table 7).

Table 7: Dose response mRNA inhibition of Eg5/KSP dupiexes in Hela cells

Concentrations given in pM [ Txcees [| [¥Cses | | JCs6s ] 1 2" ist 2nd 1st 2nd

Duplex name | screen screen screen screen screen screen

ADI2077 | 119 | 080 | 614 | 10.16 38.63 | 76.16

ADT28 | 2543

ADTNSS | 908 | 124 | 4057 257.68

Abies | 10s | 097 | om | _4s4 | 0031 | 0778] 3014] 4908

ADDIS | 0@ | 04 | 3% | 3% | mas | 4s 3121

ADL2151 7.56 | 867.36 | 66.69 | 1316527 ND

AD12157 2633

AD12166

ADI12189 28.85 28.85 101.06 101.06 847.21 847.21

AD12185 13.91 109.80 | 120.63

AD12194 5.37 5.09 53.03 | 3092 151173 | 1893 | 3674

AD12257 2.68 | 22.75 | 124.69 | 13582

AD12280 20.23 64.80 | 104.82

Avi 1901 | 35.08

AD12282 456.42 | 20.09 | 558.00

ADI12285 128 | 357.30 261.79 | 42.53

AD12292 | 40.23 | 12.00 | 929.11 | 109.10

AD12252 18.63 68.24 138.09 | 404.91

AD12275 25.04 | 123.89 | 133.10 | 1054.54 | 776.25

AD12266 4.85 7.86 160.00 32.94 41.67 162.65

AD12267 1.39 12.00 283.03 | 5L12

AD12264 15.12 56.36 | 196.78

AD12268 22.16 | 25.64 | 258.27 | 150.84

AD12279 28.54 | 23.19 | 9687 | 327.28 | 607.27

AD12256 138.04 | 77554 | 1076.76

AD12259 40.12 50.03 | 219.42

AD12276 | 19.49 89.60 672.51 | 736.72

ADI2321 2488 | 19.43 139.50 | 89.49 (ND-not determined)

Example 4. Silencing of liver Eg5/KSP in juvenile rats following single-bolus administration of LNPO1 formulated siRNA

From birth until approximately 23 days of age, Eg5/KSP expression can be detected in the growing rat liver, Target silencing with a formulated Eg5/KSP siRNA was evaluated in juvenile rats using duplex AD-6248..

KSP Duplex Tested

Duplex IID Target Sense Antisense

ADG243 KSp AccGAAGUGuuGUuuGneeTsT (SEQ ID NO:1238 } GGACAAACAACACUUCGGUTST {SEQ ID NO:1239)

Methods

Dosing of animals. Male, juvenile Sprague-Dawley rats (16 days old) were administered single doses of lipidoid (“LNPO1™) formulated siRNA via tail vein injection.

Groups of ten animals received doses of 10 milligrams per kilogram (mg/kg) bodyweight of either AD6248 or an unspecific siRNA. Dose level refers to the amount of siRNA duplex administered in the formulation. A third group received phosphate-buffered saline. Animals were sacrificed two days after siRNA administration. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.

mRNA measurements. Levels of Eg5/KSP mRNA were measured in livers from all treatment groups. Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase XK. Levels of Eg5/KSP and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for EgS/KSP were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment,

Statistical analysis. Significance was determined by ANOVA followed by the Tukey post-hoc test.

Results

Data Summary

Mean values (standard deviation) for EgS/KSP mRNA are given. Statistical significance (p value) versus the PBS group is shown (ns, not significant [p>0.05]).

Table 8. Experiment 1

KSP/GAPDH p value

PBS LO=(A7

ADO248 10 mg/kg 0.47+0.12 <(.061

Unspec 10 mg/kg 1040.26 ns

A statistically significant reduction in liver EgS/KSP mRNA was obtained following treatment with formulated AD6248 at a dose of 10 mg/kg.

Example 5. Silencing of rat liver VEGF following intraveneus infusion of LNPO1 formulated VSP

A “lipidoid” formulation comprising an equimolar mixture of two siRNAs was administered to rats. As used herein, VSP refers to a composition having two siRNAs, one directed to Eg5/KSP and one directed to VEGF. For this experiment the duplex AD3133 directed towards VEGF and AD12115 directed towards Eg5/KSP were used. Since Eg5/KSP expression is nearly undetectable in the adult rat liver, only VEGF levels were measured following siRNA treatment. siRNA duplexes administered (VSP)

Duplex iD Target Sense Antisense ucGAGAAUcuARAcuRACuTsT | AGUUAGUUUAGATUCUCGATST

AD12115 | BEg5/ESP | (SEQ ID NO:1240) (SEQ ID NO: 1241)

GCACAUAGGAGAGAUGAGCUsU | AAGCUcCAUCUCUCCuAuGuGCusG

AD3133 | VEGF (SEQ ID NO:1242) (SEQ ID NO:1243)

Key: A,G,C,U-ribonucleotides; ¢,u-2"-0O-Me ribonucleotides; s-phosphorothioate.

Unmodified versions of each strand and the targets for each siRNA are as follows unmod antisense | 3’ TTAGUCCUUAGAUUUGAUUGA 5 SEQ ID MO:1535

EgS/KSP | target 5 UCGAGARUCUAARCUAACU 3 SEQ ID NO:1311

REE 1]

VEGF | unmod antisense | 3’ GUCGUGUAUCCUCUCUACUCGAA 5 SEQ ID NO:1537 target 5 GCACAUAGGAGAGAUGAGCUU 3° SEQ ID NO:1538

Methods

Dosing of animals. Adult, female Sprague-Dawley rats were administered lipidoid (“LNP01”) formulated siRNA by a two-hour infusion into the femoral vein. Groups of four animals received doses of 5, 10 and 15 milligrams per kilogram (mg/kg) bodyweight of formulated siRNA. Dose level refers to the total amount of siRNA duplex administered in the formulation. A fourth group received phosphate-buffered saline. Animals were sacrificed 72 hours after the end of the siRNA infusion. Livers were dissected, flash frozen in liquid

Nitrogen and pulverized into powders.

Formulation Procedure

The lipidoid ND98-4HCl (MW 1487) (Formula 1, above), Cholesterol (Sigma-

Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared: ND98, 133 mg/mL;

Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. ND98, Cholesterol, and PEG-

Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted.

Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.

Characterization of formulations

Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection.

They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated sIRNA is incubated with the RNA- binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total SIRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at feast 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm, The preferred range is about at least 50 am to about at least 110 nm, preferably about at least 60 nm to about at least 100 nm, most preferably about at least 80 nm to about at least 90 nm. In one example, each of the particle size comprises at least about 1:1 ratio of Eg5 dsRNA to VEGF dsRNA. mRNA measurements. Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of VEGF and

GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched

DNA assay (GenoSpectra). Mean values for VEGF were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.

Protein measurements. Samples of each liver powder (approximately 60 milligrams) were homogenized in 1 ml RTPA buffer. Total protein concentrations were determined using the Micro BCA protein assay kit (Pierce). Samples of total protein from each animal was used to determine VEGF protein levels using a VEGF ELISA assay (R&D systems). Group means were determined and normalized to the PBS group for each experiment.

Statistical analysis. Significance was determined by ANOVA followed by the Tukey post-hoc test

Results

Data Summary

Mean values (standard deviation} for mRNA (VEGF/GAPDH) and protein (rel

VEGF) are shown for each treatment group. Statistical significance (p value) versus the PBS group for each experiment is shown.

Table 9. :

VEGF/GAPDH p value rel VEGF p value

PBS 1.6=0.17 1.0£0.17 mgkg 0.74%0.12 <0.05 © 0.2320.03 <{.001 mg/kg (3.6540.12 <(.005 0.2240.03 <(.001 mgrkg (1.49017 <0.001 0.20+£0.04 <0.001

Statistically significant reductions in liver VEGF mRNA and protein were measured at all three siRNA dose levels,

Example 6. Assessment of VSP SNALP in mouse models of human hepatic tumors.

These studies utilized a VSP siRNA cocktail containing dsRNAs targeting KSP/Eg5 and dsRNAs targeting VEGF. As used herein, VSP refers to a composition having two siRNAs, one directed to EgS/KSP and one directed to VEGF. For this experiment the duplexes AD3133 (directed towards VEGF) and AD12115 (directed towards Eg5/KSP) were used. The siRNA cocktail was formulated in SNALPs,

The maximum study size utilized 20-25 mice. To test the efficacy of the siRNA

SNALP cocktail to treat liver cancer, 1x1076 tumor cells were injected directly into the left lateral lobe of test mice. The incisions were closed by sutures, and the mice allowed to recover for 2-5 hours. The mice were fully recovered within 48-72 hours. The SNALP siRNA treatment was initiated 8-11 days after tumor seeding.

The SNALP formulations utilized were (1) VSP (KSP + VEGF siRNA cocktail (1:1 molar ratio)); (ii) KSP (KSP + Luc siRNA cocktail); and (iit) VEGF (VEGF + Luc siRNA cocktail). All formulations contained equal amounts (mg) of each active siRNA. All mice received a total sIRNA/lipid dose, and each cocktail was formulated into 1:57 cDMA

SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug using original citrate buffer conditions.

Human Hep3B Study A: anti-tumor activity of VSP-SNALP

Human Hepatoma Hep3B tumors were established in scid/beige mice by intrahepatic seeding. Group A (n=6) animals were administered PBS; Group B (n=6) animals were administered VSP SNALP; Group C (n=35) animals were administered KSP/Luc SNALP; and

Group D (n=5) animals were administered VEGF/Luc SNALP.

SNALP treatment was initiated eight days after tumor seeding. The SNALP was dosed at 3 mg/kg total siRNA, twice weekly (Monday and Thursday), for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was administered at day 23, and the terminal endpoint was at day 27.

Tumor burden was assayed by (a) body weight; (b) liver weight; (¢} visual inspection + photography at day 27; {d) human-specific mRNA analysis; and (e) blood alpha- fetoprotein levels measured at day 27.

Table 10 below illustrates the results of visual scoring of tumor burden measured in the seeded (left lateral) liver lobe. Score: “-“= no visible tumor; “+"= evidence of tumor tissue at injection site; “++” = Discrete tumor nodule protruding from liver lobe; “4+ = large tumor protruding on both sides of liver lobe; “+++” = large tumor, multiple nodules throughout liver lobe.

Table 10.

TT Wiowe | Tumor Burden

Group A: PBS, day 27 1 H+ 2 et 3 ++ 4 fe + 6 Ft

Group B: VSP 1 + (VEGF + KSP/Eg$, d. 27 2 . 3 - 4 - 5 ob 6 -

Group C: KSP 1 + (Luc + KSP), d. 27 2 ++ 3 - 4 + 5 ++

Group D: VEGF 1 Att (Luc + VEGF), d. 27 2 - 3 BR 4 At 5 Ft

Liver weights, as percentage of body weight, are shown in FIG. 1.

Body weights are shown in FIGs. 2A-2D. .

From this study, the following conclusions were made. (1) VSP SNALP demonstrated potent anti-tumor effects in Hep3B 1H model; (2) the anti-tumor activity of the

VSP cocktail appeared largely associated with the KSP component; (3) anti-KSP activity was confirmed by single dose histological analysis; and (4) VEGF siRNA showed no measurable effect on inhibition of tumor growth in this model.

Human Hep3B Study B: prolonged survival with VSP treatment

In a second Hep3B study, human hepatoma Hep3B tumors were established by intrahepatic seeding into scid/beige mice. These mice were deficient for lymphocytes and natural killer (NK) cells, which is the minimal scope for immune-mediated anti-tumor effects.

Group A (n=6) mice were untreated; Group B (n=6) mice were administered luciferase (luc) 1955 SNALP (Lot No. AP10-02); and Group C (n=7) mice were administered VSP SNALP (Lot No. AP10-01). SNALP was 1:57 cDMA SNALP, and 6:1 lipid:drug.

SNALP treatment was Initiated eight days after tumor seeding. SNALP was dosed at 3 mg/kg siRNA, twice weekly (Mondays and Thursdays), for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was delivered at day 25, and the terminal endpoint of the study was at day 27.

Tumor burden was assayed by (1) body weight; {2) visual inspection + photography at day 27; (3) human-specific mRNA analysis; and (4) blood alpha-fetoprotein measured at day 27.

Body weights were measured at each day of dosing (days 8, 11, 14, 18, 21, and 25) and on the day of sacrifice (FIG. 3).

Table 11.

Mouse | Tumor Burden by macroscopic observation

Group A: untreated, AIR Es day 27 AlG a

ATW -

AZR +t

A2G +++

AW At

Group B: BIR free 1985 Luc SNALP, day 27 B1G +++

BIW dd

B2R ++

B2G perf

B2W et

Group C; CIR -

VSP SNALP, day 27 CiG -

CiB -

CiW +

CIR +

C2G +

C2W -

Score; “= no visible tumor; “+= evidence of tumor tissue at injection site; “+H =

Discrete tumor nodule protruding from liver lobe; “+++” = large tumor protruding on both sides of liver lobe; “4++++" = large tumor, multiple nodules throughout liver lobe.

The correlation between body weights and tumor burden are shown in FIGs. 4, 3 and .

A single dose of VSP SNALP (2 mg/kg) to Hep3B mice also resulted in the formation of mitotic spindles in liver tissue samples examined by histological staining. - Turner burden was quantified by quantitative RT-PCR (pRT-PCR) (Tagman).

Human GAPDH was normalized to mouse GAPDH via species-specific Taqman assays.

Tumor score as shown by macroscopic observation in the table above correlated with

GADPH levels (FIG. 7A).

Serum ELISA was performed to measure alpha-fetoprotein (AFP) secreted by the tumor. As described below, if levels of AFP go down after treatment, the tumor is not growing. Treatment with VSP lowered AFP levels in some animals compared to treatment with controls (FIG. 7B).

Human HepB3 Study C:

In a third study, human HCC cells (HepB3) were injected directly into the liver of

SCID/beige mice, and treatment was initiated 20 days later. Group A animals were administered PBS; Group B animals were administered 4 mg/kg Lue-1955 SNALP; Group C animals were administered 4 mg/kg SNALP-VSP; Group D animals were administered 2 mg/kg SNALP-VSP; and Group E animals were administered 1 mg/kg SNALP-VSP.

Treatment was with a single intravenous (iv) dose, and mice were sacrificed 24 hr, later.

Tumor burden and target silencing was assayed by qRT-PCR (Tagman), Tumor score was also measured visually as described above, and the results are shown in the following table, hGAPDH levels, as shown in FIG. 8, correlates with macroscopic tumor score as shown in the table below,

Table 12. observation

A3 +t

Ad ret

Group B: 4 mg/kg Luc- Bl +

B3 +++

B4 : ++

Group C: 4 mg/kg Ci +

SNALP-VSP C2 ++

C3 ++

C4 +++

Group D: 2 mg/kg D1 ++ :

SNALP-VSP D2 +

D3 +

D4 ++

Group E: 1 mg/kg El +++

SNALP-VSP E2 +

E3 ++

E4 +

Score: “+= variable tumor take/ some small twmors; “++ = Discrete tumor nodule protruding from liver lobe; “+++” = large tumor protruding on both sides of liver lobe

Human (tumor-derived) KSP silencing was assayed by Tagman analysis and the results are shown in FIG. 10. hKSP expression was normalized to hGAPDH. About 80% turnor K.SP silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at I mg/kg. The clear bars in FIG. 9 represent the results from small (low GAPDH) tumors.

Human {tumor-derived} VEGF silencing was assayed by Tagman analysis and the results are shown in FIG. 10. hVEGF expression was normalized to hGAPDH. About 60% tumor VEGF silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at mg/kg. The clear bars in FIG. 10 represent the results from small (low GAPDH) tumors.

Mouse (liver-derived) VEGF silencing was assayed by Taqman analysis and the results are shown in FIG. 11A. mVEGF expression was normalized toc hGAPDH. About 50% liver VEGF silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at 1 mg/kg.

Human HepB3 Study D: contribution of each dsRNA to tumor growth

Tn a fourth study, human HCC cells (HepB3) were injected directly into the liver of

SCID/beige mice, and treatment was initiated 8 days later. Treatment was with intravenous (iv) bolus injections, twice weekly, for a total of six does. The final dose was administered at day 25, and the terminal endpoint was at day 27.

Tumor burden was assayed by gross histology, human-specific mRNA analysis (hGAPDH PCR), and blood aipha-fetoprotein levels (serum AFP via ELISA),

In Study 1, Group A was treated with PBS, Group B was treated with SNALP-

KSP+Luc (3 mg/kg), Group C was treated with SNALP-VEGF+Luc (3 mg/kg), and Group D was treated with ALN-VSP02 (3 mg/kg).

In Study 2, Group A was treated with PBS; Group B was treated with SNALP-

KSP+Luc {1 mg/kg), Group C was treated with ALN-VSPO2 (1 mg/kg).

Both GAPDH mRNA levels and serum AFP levels were shown to decrease after treatment with ALN-VSP02 (FIG. 11B).

Histology Studies:

Human hepatoma Hep3B tumors were established by intrahepatic seeding in mice.

SNALP treatment was initiated 20 days after tumor seeding. Tumor-bearing mice (three per group) were treated with a single intravenous (IV) dose of (i) VSP SNALP or (if) control (Luc) SNALP at 2 mg/kg total siRNA.

Liver/tumor samples were collected for conventional H&E histology 24 hours after single SNALP administration.

Large macroscopic turnor nodules (5-10 mum) were evident at necroscopy.

Effect of ALN-VSP in Hep3B mice:

ALN-VSP (a cocktail of KSP dsRNA and VEGF dsRNA) treatment reduced tumor burden and expression of tumor-derived KSP and VEGF. GAPDH mRNA levels, a measure of tumor burden, were also observed to decline following administration of ALN-VSP dsRNA (see FIGs. 12A-12C). A decrease in tumor burden by visual macroscopic observation was also evident following administration of ALN-VSP.

A single IV bolus injection of ALN-VSP also resulted in mitotic spindle formation that was clearly detected in liver tissue samples from Hep3B mice. This observation indicated cell cycle arrest.

Example 7. Survival of SNALP-VSP animals versus SNALP-Luc treated animals

To test the effect of siRNA SNALP on survival rates of cancer subjects, tumors were established by intrahepatic seeding in mice and the mice were treated with SNALP-siRNA.

These studies utilized a VSP siRNA cocktail containing dsRNAs targeting KSP/Eg5 and

VEGF. Control was dsRNA targeting Luc. The siRNA cocktail was formulated in

SNALPs.

Tumer cells (Human Hepatoma Hep3B, 1x1076) were injected directly into the left lateral lobe of scid/beige mice. These mice were deficient for lymphocytes and natural killer (NK cells, which is the minimal scope for immune-mediated anti-tumor effects. The incisions were closed by sutures, and the mice allowed to recover for 2-5 hours. The mice were fully recovered within 48-72 hours.

All mice received a total siRNA/lipid intravenous (iv) dose, and each cocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA,; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug using original citrate buffer conditions. siRNA-SNALP treatment was initiated on the day indicated below {18 or 26 days) after tumor seeding. sIRNA-SNALP were administered twice a week for three weeks after 18 or 26 davs at a dose of 4 mg/kg. Survival was monitored and animals were euthanized based on humane surrogate endpoints (e.g., animal body weight, abdominal distension/discoloration, and overail health),

The survival data for treatment initiated 18 days after tumor seeing is summarized in

Table 13, Table 14, and FIG. 13A.

Table 13. Kaplan-Meier (survival) data (% Surviving)

SNALP- SNALP-

Day Luc VSP } no

Tov 1007 00% 100%

CEL

Table 14. Survival in days, for each animal.

Treatment

Animal group Survival

SNALP-Luc

SNALP-Luc | 33

SNALP-Luc

SNALPLuc | 33 | days

I 5 | SNALPLuc | 35 | days 6 | SNALPLuc | 38 | days [7 | SNALPduc | 57 | days _8 | SNALP-vSP 9 | SNALP-VSP 51

SNALP-VSP | 51 | days _11 | SNALPVSP | 51 | days 12 | SNALPVSP | 53 | days : |_13 | SNALPVSP | 53 | days

SNALP-VSP | 57 | days

SNALP-VSP | 57

FIG. 13A shows the mean survival of SNALP-VSP animals and SNALP-Luc treated animals versus days after tumor seeding. The mean survival of SNALP-VSP animals was extended by approximately 15 days versus SNALP-Luc treated animals.

Table 15. Serum alpha fetoprotein (AFP) concentration, for each animal, at a time pre-treatment and at end of treatment {concentration in pg/ml) on bre-Rx Rx

SNALP-Luc | 10.088 | 202.082 : SNALP-Luc | 23.736 | 648.952 13,308

SNALP-Luc | 4.778 | 338.688 6 | SNALP-Luc 826.972 24501 8 | SNALP-vSP 182.35 9 | SNALP-VSP | 91516 | 24806

SNALP-VSP

SNALP-VSP 149.352

SNALP-VSP

SNALP-VSP | 4516

Tumor burden was monitored using serum AFP levels during the course of the experiment. Alpha-fetoprotein (AFP) is a major plasma protein produced by the yolk sac and the liver during fetal life. The protein is thought to be the fetal counterpart of serum albumin, and human AFP and albumin gene are present in tandem in the same transcriptional orientation on chromosome 4, AFP is found in monomeric as well as dimeric and trimeric forms, and binds copper, nickel, fatty acids and bilirubin. AFP levels decrease gradually after birth, reaching adult levels by 8-12 months. Normal adult AFP levels are low, but detectable.

AFP has no known function in normal adults and AFP expression in adults is often associated with a subset of tumors such as hepatoma and teratoma. AFP is a tumor marker used to monitor testicular cancer, ovarian cancer, and malignant teratoma. Principle tumors that secrete AFP include endodermal sinus tumor (yolk sac carcinoma}, neuroblastoma, hepatoblastoma, and heptocellular carcinoma. In patients with AFP-secreting tumors, serum levels of AFP often correlate with tumor size. Serum levels are useful in assessing response to treatment. Typically, if levels of AFP go down after treatment, the tumor is not growing.

A temporary increase in AFP immediately following chemotherapy may indicate not that the tumor is growing but rather that it is shrinking (and releasing AFP as the tumor cells die).

Resection is usually associated with a fall in serum levels. As shown in Figure 14, tumor burden in SNALP-VSP treated animals was significantly reduced.

The experiment was repeated with SNALP-siRNA treatment at 26, 29, 32 35, 39, and 42 days after implantation. The data is shown in FIG. 13B. The mean survival of SNALP-

VSP animals was extended by approximately 15 days versus SNALP-Luc treated animals by approximately 19 days, or 38%.

Example 8. Induction of Mono-asters in Established Tumors

Inhibition of KSP in dividing cells leads to the formation of mono asters that are readily observable in histological sections. To determine whether mono aster formation occurred in SNALP-VSP treated tumors, tumor bearing animals (three weeks after Hep3B cell implantation) were administered 2 mg/kg SNALP-VSP via tail vein injection. Control animals received 2 mg/kg SNALP-Luc. Twenty four hours later, animals were sacrificed, and tumor bearing liver lobes were processed for histological analysis. Representative images of H&E stained tissue sections are shown in Figure 15. Extensive mono aster formation was evident in ALN VSPO02 treated (A), but not SNALP-Luc treated (B), tumors.

In the latter, normal mitotic figures were evident. The generation of mono asters is a characteristic feature of KSP inhibition and provides further evidence that SNALP-VSP has significant activity in established liver tumors,

Example 9. Manufacturing Process and Product specification of ALN-VSP02 (SNALP-VSP)

ALN-VSPO2 product contains 2 mg/mL of drug substance ALN-VSPDS01 formulated in a sterile lipid particle formulation (referred to as SNALP) for IV administration via infusion. Drug substance ALN-VSPDS01 consists of two siRNAs (ALN-12115 targeting

KSP and ALN-3133 targeting VEGF) in an equimolar ratio. The drug product is packaged in mL glass vials with a fill volume of 5 mL.

The following terminology is used herein: ee ET Sores A-19562 * Alternate names = AD-12115, AD121135; ¥* Alternate names = AD-3133, AD3133 9.1 Preparation of dmg substance ALN-VSPDS01

The two siRNA components of drug substance ALN-VSPDS0I, ALN-12115 and

ALN-3133, are chemically synthesized using commercially available synthesizers and raw materials, The manufacturing process consists of synthesizing the two single strand oligonucleotides of each duplex (A 19562 sense and A 19563 anfisense of ALN 12115 and A 3981 sense and A 3982 antisense of ALN 31 33) by conventional solid phase oligonucleotide synthesis using phosphoramidite chemistry and 5° O dimethoxytriphenylmethyl (DMT) protecting group with the 2” hydroxyl protected with tert butyldimethylsilyl (TBDMS) or the 2” hydroxyl replaced with a 2° methoxy group (2° OMe). Assembly of an oligonucleotide chain by the phosphoramidite method on a solid support such as controlled pore glass or polystyrene. The cycle consists of 5” deprotection, coupling, oxidation, and capping. Each coupling reaction is carried out by activation of the appropriately protected ribo , 2° OMe, or deoxyribonucleoside amidite using 5 {ethylthio) 1H tetrazole reagent followed by the coupling of the free 5° hydroxyl group of a support immobilized protected nucleoside or oligonucleotide. After the appropriate number of cycles, the final 5° protecting group is removed by acid treatment. The crude oligonucleotide is cleaved from the solid support by aqueous methylamine treatment with concomitant removal of the cyanoethyl protecting group as well as nucleobase protecting groups. The 2° O TBDMS group is then cleaved using a hydrogen fluoride containing reagent fo yield the crude oligoribonuclectide, which is purified using strong anion exchange high performance liquid chromatography (HPLC) followed by desalting using ultrafiltration. The purified single strands are analyzed to confirm the correct molecular weight, the molecular sequence, impurity profile and oligonucleotide content, prior to annealing into the duplexes. The annealed duplex intermediates ALN 12115 and ALN 3133 are either lyophilized and stored at 20°C or mixed in 1:1 molar ratio and the solution is lyophilized to yield drug substance ALN VSPDSO01. If the duplex intermediates were stored as dry powder, they are redissolved in water before mixing. The equimolar ratio is achieved by monitoring the mixing process by an HPLC method.

The manufacturing process flow diagram is shown in Figure 16.

Example specfications are shown in Table 16a.

The results of up to 12 month stability testing for ALN-VSPDS0]1 drug substance are shown in Tables 16c. The assay methods were chosen to assess physical property (appearance, pH, moisture), purity (by SEC and denaturing anion exchange chromatography) and potency (by denaturing anion exchange chromatography [AX-HPLC]).

Table 16a. Example specifications for ALN-VSPDSGO1

Method Acceptance Criteria

White to off-white powder identity, ALN-VSPDS01 Duplex AX-HPLC Dupiex retention fimes are consistent

ALN-3133 with those of reference standards

ALN-12118 identity, ALN-VSPDS01 MS Molecular weight of single strands are within the following ranges:

A-3581: 6869-6873 Da

A-3982; 7305-7309 Da

A-189562: 6762-6766 Da

A-19563: 6675-8679 Da

Sodium counter ion (Y%ew/w on Flame AAS or ICP-0ES Report data anhydrous basis

ALN-VSPDS01 assa Denaturing AX-HPLC 80 — 110%

Purity of ALN-VSPDSD1 = 90.0 area %

Single strand purity, Denaturing AX-HPLC Report data

ALN-VSPDS01 Report area % for total impurities

SIRNA molar ratio Duplex AX-HPLC 1.0 +01

Moisture content Karl Fischer titration

Residual solvents HS-Capillary GC

Acetonitrite } £410 ppm

Ethanol = 5000 ppm ] |sopropancl < 5000 ppm oH of 1% seiution USP <791>

Heavy metals ICP-MS Report data

As, Cd, Cu, Cr, Fe, Ni, Pb, Sn

Bacterial endotoxins USP <85> = 0.5 EU/mg

Modified USP <61> < 100 CFU/g

Table 16h: Stability of drug substance

Study Storage Conditions: -20°C (Storage Condition) } Acceptance white powder ‘pH TUSP<791> | Reporidata | 67 [64 166 [64 [68

Moisture oa : content al Fischer < 15% 3.6% 6.7 6.2 5.6 5.0 i (owiw :

A-3981 oo (area % =~

A-3982 (antisense) Denuring AX | Report data 23 24 (area %

A19562 : (area %}

A-19553 — (area %) 9.2 Preparation of drug product ALN-VSPO2 (SNALP-VSP)

ALN VSP02, is a sterile formulation of the two siRNAs (in a 1:1 molar ratio) with lipid excipients in isotonic buffer. The lipid excipients associate with the two siRNAs, protect them from degradation in the circulatory system, and aid in their delivery to the target tissue. The specific lipid excipients and the quantitative proportion of each (shown in Table 17) have been selected through an iterative series of experiments comparing the physicochemical properties, stability, pharmacodynamics, pharmacokinetics, toxicity and product manufacturability of numerous different formulations. The excipient DLinDMA is a titratable aminolipid that is positively charged at low pH, such as that found in the endosome of mammalian cells, but relatively uncharged at the more neutral pH of whole blood. This - feature facilitates the efficient encapsulation of the negatively charged siRNAs at low pH,

P g p preventing formation of empty particles, yet allows for adjustment (reduction) of the particle charge by replacing the formulation buffer with a more neutral storage buffer prior to use.

Cholesterol and the neutral lipid DPPC are incorporated in order to provide physicochemical stability to the particles. The polyethylenegiycol lipid conjugate PEG2000 C DMA aids drug product stability, and provides optimum circulation time for the proposed use. ALN

VSP(2 lipid particles have a mean diameter of approximately 80-90 nm with low polydispersity values. A representative cryo transmission electron microscope {cryo TEM) image is shown in Figure 17. At neutral pH, the particles are essentially uncharged, with

Zeta Potential values of less than 6 mV. There is no evidence of empty (non loaded) particles based on the manufacturing process.

Table 17: Quantitative Composition of ALN-VSP0O2 © Componentgrade | oo ‘Component, | Proportion (mg/ml) | Proportion (mg/mL) To : eowomow | ar

DLinDMA (1,2-Dilinoleyloxy- "3 ; : N,N-dimethyi-3-aminopropane), ’ i cGMP

DPPC (R-1,2-Dipalmitoyl-sn-glycero-3- 1 phosphocholine}, cGMP ’ i Cholesterol, Synthetic, cGMP 2

PEG2000-C-DMA (3-N-[{-Methoxy poly(ethylene glycol) 2000)

So . 0.8 carbamoyi}-1,2-dimyristyloxy-propylamine}, cGMP

Phosphate Buffered Saline, cGMP * The 1:1 molar ratio of the two siRNAs in the drug product is maintained throughout the size distribution of the drug product particles.

Solutions of lipid (in ethanol) and ALN VSPDS01 drug substance (in aqueous buffer) are mixed and diluted to form a colloidal dispersion of siRNA lipid particles with an average particle size of approximately 80-90 nm. This dispersion is then filtered through 0.45/0.2 um filters, concentrated, and diafiltered by Tangential Flow Filtration. After in process testing and concentration adjustment to 2.0 mg/mL, the product is sterile filtered, aseptically filled into glass vials, stoppered, capped and placed at 5 = 3°C. The ethanol and all aqueous buffer components are USP grade; all water used is USP Sterile Water For Injection grade.

Representative ALN-VSPO2 process is shown in flow diagram in FIG. 18.

Table 18a; Example ALN-VSP0O2 specifications

Analytical Procedure Acceptance Criteria

White to off-white, homogeneous

Appearance Visual | opalescent liguid, no foreign particles

USP <791> 68-78

Osmciali USP <785> 250 — 350 mOsm/kg dently, ALN-VSPD S01 Duplex Retention times consistent with

ALN-12115 Anion Exchange (AX)-HPLC those of reference siandards

Identity, ALN-VSPD301

A-3981 n , Rr on fi , .

A-3082 aud retention imes consistent with

A-19562 - those of reference standards

A-19583

Lipid identity

DLInBMA Reversed Phase (RP)-HPLC with ow . .

PEG s50-C-DMA Evaporative Light Scattering Retention times consistent in

DPPC (ELS) detection

Cholesterol

ALN-VSPDS01 label claim Denaturing

Strength/Potency) AX-HPLC 1.7- 2.3 mg/ml ; Dupiex

Duplex molar ratio AX-HPLE 1.0% 0.1 . RP-HPLC with

DLInDMA conient ELS detection 5.6- 10.3 mg/mL

RP-HPLC with

PEG op00-C-DMA content £1 8 detection 0.61.1 mgmt

RP-HPLC with

DPPC content ELS detection 0.8 - 1.5 mg/mL

RP-HPLC with

Cholestero! content ELS detection 2.1- 3.9 mgimL

Calculated from total lipid assay

Total lipid: ALN-VSPDS01 ratio and label claim for drug 4.8 — 8.1 mg/mg ] substance

ALN-VSPDS01 encapsulation = 20.0% . Denaturing o

AX-HPLC > 80.0 area % . Report retention times (relative to impurity profile Denaturing A-19583) and area % for alt peaks

AX-HPLC a 2 0.20%

Residual ethanol USP <467> < 5000 ppm ) jon Pairing {IP}-HPLC with UV

Residual EDTA detection 5 2000 ug/mi

Particle size Z average Dynamic light scattering 80-120 nm

Polydispersit Dynamic light scattering

Particle size distribution : oe Dynamic fight scattering Report data i Deo

Particulate matter . : 2 25 ym Modified USP <788> | = 300 per container > 10 um % 3000 per container

Bacterial endotoxing Modified USP <85> < 5.0 EU/mL

Sterilit USP <71>

Volume in container USP <i>

USP <905> Pass

Inductive Coupled Plasma Mass

Heavy metal analysis Spectrometry Report data {ICP-MS

9.4 Container/Closure Svstem

The ALN VSPO02 drug product is packaged in 10 mL glass vials with a fill volume of mL. The container closure system is comprised of a USP/EP Type 1 borosilicate glass vial, a teflon faced butyl rubber stopper and an aluminum flip off cap. The drug product will be stored at 5 + 3°C, 9.5 Stability of drue product ALN-VSP0O2

Stability data (25°C/60%RH) are given in Table 18b and 18c.

Table 18b: Example ATN-VSP02 stability at storage conditions

Lot Ne.: IC097 Study Storage Conditions: 2-8°C rcoent

Test Method oo ance 1 3 3 4 6 riteria Initial :

Month | Months | Months | Months | Months

While 10 off-white,

Appearanc " homogeneous a e Visual opalescent liquid, Pass Pass Pass Pass Pass ‘no foreign particles .

TEE

ALN-

VSPDS01 Retention times {dentity, Duplex consistent with

ALN-3133 | AXHPLC | those of reference | L055 | Pass Pass Pass | Pass

AlLN- standards 12115

ALN- .

VSPDSO1 Co identity, Denaturin Retention times

A-3981 g Tose. of reference Pass Pass Pass Pass Pass Pass

A-3982 AX-HPLC siandards

A-19582 i A-19563

Lipid identity, i DLinDMA | Retention times i RP-HPLC . Co

PEG000- with ELS consistent with Pass Pass Pass Pass Pass | Pass

C-DMA Detect those of reference

DPPC etection standards

Cholestero

ALN- .

Denaturin otenc AXHPLL

EE | ee wl we ew molar ratio | AXHPLG 1.0+0G1 1.0 1.0 1.0 1.0 , RP-HPLC

Detection

RP-HPLC

Cholesiero | ue) 21-39mg/ml | 3.4 3.5 3.4 3.5 3.4 3.5 content Detection

Lot No.: IC097 Study Storage Conditions: 2-8°C —

Test Method on ance i 2 3 4 6 riteria Initial

Month | Months | Months | Months | Months

RP-HPLC

Detection .

PEGo0~ RP-HPLC

C-DMA with ELS 0.6-1.1 mg/mL 1.0 1.0 1 1.0 1.0 content Detection j

Total lipid: ALN- | Calculatio

VSPDSO1 | n 4.9 ~ 8.1 mg/mg 7.0 6.9 7.1 7.4 7.0 7.1 ratio

ALN-

VSPDS01 | Fiuoromet >90.0% 959 | 965 | 944 | 98.1 97.8 96.4 encapsulat | ric assay ion :

Denaturin

Purity g = 80.0% 90.7 89.6 91.3 92.4 90.8

AX-HPLC

Pariicle . . Light “ za oom " Y v v

Z-average

Si scattering : Particle size Light distribution | scattering Report data (nm) 56 56 56 56 56 56

Do

Particle size Light distribution | scattering Report data (nm} 76 77 77 7 78 77 , Do

Particle size Light , distribution | scattering Report data (um) 110 12 112 113 112 113 , Dao

Paricuiate Modified {per container)

Sos Usp < 300 18 NS NS NS NS 3

Z2oH <788> <3000 48 1 } 10 um endotoxins

Table 18¢: Example ALN-VSP02 stability at 25°C/ambient humidity

Lot No.: ICO Study Sterage Conditions: 25°C/ambient humidity

A ; Hesults

Test Method oa ance LL. 1 2 3 4 6 riteria Initial

Menth | Months | Months | Months | Months

White to off-white,

Appearanc Visual homogeneous Pass Pass Pass Pass Pass e opalescent liquid, no foreign particles

Lot No: 1C097 Study Storage Conditions: 25°C/ambient humidity

Test Method Acceptance ; 2 3 4 6

Criteria Initial

Month | Months | Months | Months | Months

ALN-

VSPDS01 Retention times

Identity, Duplex consistent with ’ . .

ALN-3133 | AXHPLC | those ofreferonce | 25S | Dass | Pass | Pass) Pass | Pass

ALN- standards 12115

ALN- ont Retention times ! Deneturing consistent with . oe AX-HPLC {hose of reference Pags Pass Pass Pass Pass Pass " standards

A-19562

A-18563

Lipid identity,

DLIinDMA Retention times

RP-HPLC . .

PEGa0c0- with ELS consistent with Pass Pass Pass Pass Pass Pass

C-DMA Detection those of reference

DPPC standards

Cholastero

ALN-

VSPDS01 | Denaturing . strengthip AXHPLC 1.7-2.3 mg/mL 2.1 2.1 2.0 2.0 2.0 2.0 otenc

Duplex Duplex motar ratic | AX-LPLC 1.0+0.1 1.0 1.0 1.0 1.0 1-0 1.0 . RP-HPLC

DLinbMA with ELS 5.6 10.3 mg/mL 9.6 9.3 9.2 4.3 coment Detection

RP-HPLC

Detection

RP-HPLC

Detection

PEGuxe- | RP-HPLC

C-DMA with ELS 0.6 ~ 1.1 mg/mL 1.0 1.0 1.0 i.1 1.0 1.0 content Detection

Total lipid: ALN- . - 1 VSPDSo1 Calculation 4.9 8.1 mg/mg 7.0 7.3 7.4 7.6 7.4 7.5 ratio

ALN-

VSPLS0! | Fluorometri > 90.0% 959 | 97.2 94.6 91.9 97.9 96.7 encapsulat Cc assay ien we [ww ew [we | on [Te

Particie . . Z-average

Polydisper | Light <0.15 002 | 00s | 003 | 004 | 004 | 003 sity scattering

Particle size Light distrinution | scatiering Report data (nm) 36 54 56 58 56 57 , Dio oce nce

Month | Months {| Months | Months | Months

Pariicle size Light distribution | scatiering Report data (nm) 76 75 77 79 77 78 » Dso

Particle size Light distribution | scatiering Report data (nm) 110 110 111 113 LHi3 » Dog orien ae Modified {per container)

S25 um | USP <T88> =n J ¢ NS NS NS NS L > 10 um ~

Bacterial

Example 10. In Vitro Efficacy of ALN-VSP02 in Human Cancer Cell Lines

The efficacy of ALN-VSP02 treatment in human cancer cell lines was determined via measurement of KSP mRNA, VEGF mRNA, and cell viability after treatment. IC50 (nM) values determined for KSP and VEGF in each cell line.

Table 19: cell lines

Cell line tested ATCC cat number

HELA "ATCC Cat N: CCL-2

KB ATCC Cat N: CCL-17

HEP3B ATCC Cat N: HB-8064

SKOV-3 © ATCC Cat N: HTB-77

HCT-116 ATCC Cat N: CCL-247

HT-29 ATCC Cat N: HTB-38

PC-3 ATCC Cat N: CRL-1435

A549 ATCC Cat N; CCL-185

MDA-MB-231 ATCC Cat N; HTB-26

Cells were plated in 96 well plates in complete media at day 1 to reach a density of 70% on day 2. On day 2 media was replaced with Opti-MEM reduced serum media (Invitrogen Cat N: 11058-021) and cells were transfected with either ALN-VSPO2 or control

SNALP-Luc with concentration range starting at 1.8 uM down to 10 pM. After 6 hours the media was changed to complete media. Three replicate plates for each cell line for each experiment was done.

Cells were harvested 24 hours after transfection. KSP levels were measured using bDNA; VEGF mRNA levels were measured using human TaqMan assay.

Viability was measured using Cell Titer Blue reagent (Promega Cat N: G8080) at 48 and/or 72h following manufacturer’s recommendations.

As shown in Table 26, nM concentrations of VSP02 are effective in reducing expression of both KSP and VEGF in multiple human cell lines. Viability of treated cells was not

Table 20: Results TICS0 (uM) | IC50 (nM)

Cell line KSP VEGF

SKOV-3

FCTL6

Tso [Wo

Example 11. Anti-tumor efficacy of VSP SNALP vs. Sorafenib in established

Hep3B intrahepatic tumors

The anti-tumor effects of muilti-dosing VSP SNALP verses Sorafenib in scid/beige mice bearing established Hep3B intrahepatic tumors was studied. Sorafenib is a small molecule inhibitor of protein kinases approved for treatment of hepatic cellular carcinoma (HCC).

Tumors were established by intrahepatic seeding in scid/beige mice as described herein. Treatment was initiated 11 days post-seeding. Mice were treated with Sorafenib and a control SIRNA-SNALP, Sorafenib and VSP siRNA-SNALP, or VSP siRNA-SNALP only.

Control mice were treated with buffers only (DMSO for Sorafenib and PBS for siRNA-

SNALP). Sorafenib was administered intraparenterally from Mon to Fri for three weeks, at mg/kg according to body weight for a total of 15 injections. Sorafenib was administered a minimum of 1 hour after SNALP injections. The siRNA-SNATLPS were administered intravenously via the lateral tail vein according at 3 mg/kg based on the most recently recorded body weight (10 ml/kg) for 3 weeks (total of 6 doses) on days 1, 4,7, 10, 14, and 17.

Mice were euthanized based on an assessment of tumor burden including progressive weight loss and clinical signs including condition, abdominal distenstion/discoloration and mobility.

The percent survival data are shown in FIG. 21. Co-administration of VSP siRNA-

SNALP with Sorafenib increased survival proportion compared to administration of

Sorafenib or VSP siRNA-SNALP alone. VSP siRNA-SNALP increased survival proportion compared to Sorafenib.

Example 12. In vitro efficacy of VSP using variants of AD-12115 and AD-3133

Two sets of duplexes targeted to Eg5/KSP and VEGF were designed and synthesized.

Each set included duplexes tiling 10 nucleotides in each direction of the target sites for either

AD-12115 and AD-3133.

Sequences of the target, sense strand, and antisense strand for each duplex are shown in the Table below.

Each duplex is assayed for inhibition of expression using the assays described herein.

The duplexes are administered alone and/or in combination, e.g., an EgS5/KSP dsRNA in combination with a VEGF dsRNA. In some embodiments, the dsRNA are administered in a

SNALP formulation as described herein.

Table 21: Sequences of dsRNA targeted to VEGF and Eg5/KSP (tiling) on SEQ Sense Strand SEQ gene > oo NO: 50 to 3 BO: oy AcCAAGGCCAGCACAURGET ST 0447.1 | VEGTA | ACCRAGGCCAGCACRUAGS | 2264 = : CCUAUGUGCUGGCCUUGGUTST 2305

AD- CeBAGEecAGCACAUAGEAT ST 2308 2044s 1 | VEGFR | CCAAGGCCAGCACAUAGGA | 2265 : UCCuAUGUGCUGGCCUUGET TF 2307

AD- CCAAGGCCAGCACAUAGGAT ST 2308

S04sg.1 | VEGFA | CCAAGGCCAGCACAUAGGA | 2266 : CUCCUAUGUGCUGGCCUUGT ST 2309

AD~ AAGGCCAGCACAVAGGAGAT ST 0450.1 | VEGFA | BAGGCCAGCRCAURGGAGR | 2267 : UCUCCUATGUGCUGECCTUT ST

AD- ! BGG CCRGCACAVAGERGALT ST

Spasy 1 | VEGFA | AGGCCAGCACAUAGGAGRG | 2268 : CUCUCCuAUGUGCUGGCCUTST 2313

AD- GGCCAGCACARAGCAGAGATST 2314

Sass .1 | VEGFR | GGCCAGCACAURGGRGAGR | 2269 : JCUCUCCRAUGUGCUGGCCT sT oe ET near meen SEG Sense Strand SEQ

Duplex ID carget Serer sequence in Antisense strand ip g = KO: 57 to 3 NOD: mE Tas an- cCAGCACAUAGGAGAGAUGT ST 2318 204541 VEGFA | CCAGCACAUAGGAGAGRUG 2271 - cAUCUCUCCUAUGUGCUGGT ST 2319

AD- CRAGCACARUAGGAGAGAUGAT ST 2320 20455 1 VEGF2 | CAGCACAUAGGAGAGAUGA 2272 : UcAUCUCUCCuAUGUGCUGT ST 2321

Ap- AGCACAUAGGRGAGAUGAGT ST 2322 20456. 1 VEGFA | AGCACAUAGGAGAGAUGAG 2273 . CUcRUCUCUCCUAUGUGCUT ST 2323

AD- cAcAuAGGAGAGAUGAGCUTST 2324 20457. 1 VEGFA | CACAUAGGAGAGAUGAGCU 2274 : AGCUcRAUCUCUCCuAUGUGTST 2325

Ap~ ACAUAGGAGAGRUGAGCuuTsT | #326 20458. 1 VEGEA | ACAUAGGAGAGAUGAGCUU 2275 ’ ARGCUCAUCUCUCCuAUGUTST 2327

AD- CARUAGGAGAGRUGAGCuuCTsT 2328 204591 VEGFA | CAUAGGAGAGAUGAGCUCC 2276 = . GARGCUCAUCUCUCCUAUGT ST 2329

AD- AUAGGAGAGAUGAGCuuccTsT 2330 20460.1 VEGFA | AUAGGAGAGAUGAGCUUCC 2277 . GGRAGCUcAUCUCUCCuAUT ST 2331 nD VAGSAGAGAUGRGCUuCCUT ST 2332 204611 VEGFA | UAGGAGAGAUGAGCUTUCCU 2278 : AGGAAGCUcAUCUCUCCUATST 2333

AD- AGGAGAGAUGAGCUUCCUATST 2334 20462.1 VEGFA | AGGAGAGAUGAGCUUCCUA 227% = : UAGGAAGCUCAUCUCTUCCUT ST 2335

AD- CEAGAGRUGAGeuuccuAeTsT | 2336 20463.1 VEGFA | GGAGAGAUGAGCUUCCUAC 2280

GuAGGAAGCUCAUCUCUCC TST 2337 i

AD ’ GAGAGRAUGAGCRUCCUACAT ST 2338 20464.1 VEGFA | GAGAGAUGAGCUUCCUACA 2281 - UGUAGGAAGCUCAUCUCUCTsT 2339 :

AD AGAGRUGAGCUUCCURCAGTST 2340 20465. 1 VEGFA | AGAGRAUGAGCUUCCUACAG 2282 : - CUGuAGGAAGCUCAUCUCUT sT 2341 t

AD- | GAGAUGAGCUUCCUACAGCTST 2342 0466.1 VEGFA | GAGRUGAGCUUCCUACAGC 2283 . GCUGUAGGARGCUCAUCUCT ST 2343 an- AuGuuccuuducGAGAAUCT ST 2344 0467.1 KSPp AUGUUCCUUAUCGAGAALC 2284 * GAUUCUCGALARGGAACAUT ST 2345

. = SEQ Sense Strand SEQ pup one Bes

J : HO: 5° to 3 NO: mE Tan

AD- GuuccuuAucGAGAAUCUATST 2348 20469 .1 ESP GUUCCUJAUCGAGAAUCUA 2286 - UAGAUUCUCGRAUARGGRAACT ST 2349

AD- veccRuACGAGRAUCUAATST 2350 50470. 1 ESP UUCCUUAUCGAGAAUCUAA 2287 : UuAGAUUCUCGAUAAGGAAT ST 2351

Ap uccuuAncGAGAAUCUARAAT ST 2352 204711 KSP UCCUUAUCGAGAAUCUARA 2288 : UUUAGAUUCUCGABAAGGAT ST 2353

AD cCuVAUCGAGARUCRARACTST 2354 50472. 1 KSP CCUUAUCGAGAAUCUAAAC 2289 : GUUUAGAUUCUCGAUARAGGETST 2355

AD~- cuuAucGaGarucuiihculstT 2356 20473.1 KsP CUUAUCGAGARUCUAAACT 2290 : AGUUuAGAUUCUCCAUAAGT ST 2357 an- UUAUCGAGAAUCUAAACUAT ST 2358 204741 K3P UUAUCGAGAAUCUARACUA 2291 ’ VAGUUBAGAUUCUCGAUAATST 2359

AD- uAUCGAGAAUCURAACURAT ST 2360 202475. 1 KSP UAUCGAGAAUCUAAACUAA 2292 : UnAGUUuAGAUUCUCGAUAT ST 2361

I AucGAGARuCUAAACUAACTST 2362 20476. 1 KSP AUCGAGRAUCUARACURAC 2293 : GUUAGUUUAGAUUCUCGAUT ST 2363

AD- CGAGARUCUARACUAACUAT ST 2364 20477. 1 ESP CGAGAAUCUAAACURACUA 2294 " uAGUUAGUUuAGAUUCUCGT ST 2365

AD GAGAAUCUABACUAACUAGT ST 2366 204781 ESP GAGRAUCURAACURACUAG 2295 ° CuAGUUAGUUUAGAUUCUCT ST 2367

AD- AGAAUCUAAACUARCUAGATST 2368 20479. 1 KSP AGAAUCUARACULRACUAGA 22%9¢ : UCUAGURAGUUUAGATUCUT ST 236%

AD GAANCUAAACURACUAGAATST 2370 20480 .1 KSP GAAUCUAAACUARACUAGAR 2297 : UUCuAGUuAGUUuRAGAUUCT ST 2371

AD ARucCULAACURAACUAGAAUTST 2372 20481 .1 KSP ARUCUAARCUARCUAGART 2298 : AUUCHAGUuAGUULUAGADUT ST an. AucuARACuAACUAGAAUCTST 2374 20482 1 KSP AUCUARRCUAACUAGARRUC 2299 . GAUUCUAGUUAGUUUAGAUTST 2375

To le Ten

- SEQ Sense Strand SEQ 4 NO: 5' to 3° NO: me Te

AD- cuhAbcubhculGRAuccuTsT 2378

Gaps. 1 | KSP | CUARACURRCUAGAAUCCU | 2301 :- AGGAUUCUAGUUAGUURAGTST 2379

AD uAAAcuAAcuAGRAUCcUcTsT 2380 eags. 1 | KSP | URARCUARCUAGARUCCUC | 2302 : GAGGAUUCURGUUAGUUUATST | 2381

AD- AhncuihculhGhhuccucceTst 2382 ooase.y | SP | RARCUBACURGAAUCCUCC | 2303 : GGAGGAUUCUAGUUAGUUUTST | 2383

Example 13. VEGF targeted dsRNA with a single blunt end

A set duplexes targeted to VEGF were designed and synthesized. The set included duplexes tiling 10 nucleotides in each direction of the target sites for AD-3133. Each duplex includes a 2 base overhang at the end corresponding to the 3° end of the antisense strand and no overhang, e.g., a blunt end, at the end corresponding to the 5° end of the antisense strand.

The sequences of cach strand of these duplexes are shown in the following table.

Each duplex is assayed for inhibition of expression using the assays described herein.

The VEGF duplexes are administered alone and/or in combination with an EgS/KSP dsRNA (e.g., AD-12115). In some embodiments, the dsRNA are administered in a SNALP formulation as described herein.

Table 22; Target sequences of blunt ended dsRNA targeted to VEGF denlex 1D Se VEGF target sequence position on pe | - 5to3 VEGF gene

NO:

AD-20447.1 | 2384 | ACCARGGCCAGCACAUAGE

AD-20448.1 | 2385 | CCAAGGCCAGCACAUAGGA | 1366

AD-20449.1 | 2386 | CARGGCCAGCACAUAGGAG | 1367

AD-20450.1 AAGGCCAGCACAUAGGAGE | 1368

AD-20451.1 | 2388 | AGGCCAGCACAUAGGAGAG | 1369

AD-20432.1 | 2389 | GGCCAGCACAUAGGAGAGA

AD-20453.1 | 2390 | GCCAGCACAUAGGAGAGAU | 1371

AD-20454.1 {2381 | CCAGCACAUAGGAGAGAUG | 1372

AD-20455.1 CAGCACAUAGGAGAGAUGA | 1373

AD-20456.1 | 2393 | AGCACAUAGGAGAGAUGAG | 1374 aD-20457.1 | 23%4 | CACAUAGGAGAGRUGAGCU | 1376

AD-20458.1 | 2395 | ACAUAGGAGAGAUGAGCUU

AD-20459.1 | 2396 | CAUAGGAGAGAUGAGCUUC

AD-20460.1 | 2397 | AUAGGAGAGAUGAGCUUCC | 1379

AD-20461.1 | 2398 | UAGGAGAGAUGAGCUUCCU | 1380

AD-20462.1 | 2399 | AGGAGAGAUGAGCUUCCUR | 1381

: AD-20463.1 | 2400 | GGAGAGAUGAGCUUCCUAC | 1382

AD-20464.1 | 2401 | GAGAGAUGAGCUUCCURCA | 1383 : aD-20465.1 | 2402 | AGAGAUGAGCUUCCUACAG

AD-20466.1 | 2403 | GAGAUGAGCUUCCUACAGC | 1385

Table 23: Strand sequences of blunt ended dsRNA targeted to VEGF duplex ID Sense strand ee Antisense strand pe r r r ! - {3 to 3") NO: (5 to 3") —

AD-20447.1 | ACCAAGGCCAGCACAUAGGAS | 2404 | CUCCURUGUGCUGGCCUUGGUGA | 2424

AD-20448.1 | CCAAGGCCAGCACAUAGGAGA UCUCCUAUGUGCUGGCCUUGEUG | 2425

AD-20445.1 | CAAGGCCAGCACAURGGAGAG | 2406 | CUCUCCUAUGUGCUGGCCUUGGY | 2428

AD-20450.1 | AMGGCCAGCACRURGGAGAGA | 2407 | UCUCUCCUAUGUGCUGGCCUUGS | 2427 2AD-20451.1 | AGGCCAGCACAUAGGAGAGAU | 2408 | AUCUCUCCUAUGUSCUGECCUUG | 2428

AD-20452.1 | GGCCAGCACAURGGAGAGAUS | 2409 | CAUCUCUCCURUGUGCUGGCCUT | 2429

AD-20453.1 | GCCAGCACAUAGGAGAGAUGA | 2410 | UCAUCUCUCCUAUGUGCUGECCU | 2430

AD-20454.1 | CCAGCACAUAGGAGAGRUGAG | 2411 | CUCAUCUCUCCUAUGUGCUGGCC | 2431

AD-20455.1 | CAGCACAUAGGAGAGAUGAGC | 2412 | GCUCAUCUCUCCUAUGUGCUGES | 2432

AD-20456,1 | AGCACAUAGGAGAGAUGAGCU | 2413 | AGCUCAUCUCUCCUAUGUGCUGG | 2433

AD-20457.1 | CACAUAGGAGAGAUGAGCUUC | 2414 | GRAGCUCAUCUCUCCUAUGUGCT | 2434

AD-20458.1 | ACAUAGGAGAGAUGAGCUUCC | 2418 | GGAAGCUCAUCUCUCCUAUGUGC | 2435

AD-20459.1 | CAUAGGAGAGAUGAGCUUCCU | 2416 | AGGARGCUCAUCUCUCCURAUGUG | 2436

AD-20460.1 | AUAGGAGAGAUGAGCUUCCUR | 2417 | UAGGAAGCUCAUCUCUCCUAUGU | 2437

AD-20461.1 | UAGGAGAGAUGAGCUUCCUAC | 2418 | GUAGGAAGCUCAUCUCUCCUAUG | 2438

AD-20462.1 | AGGAGAGAUGAGCUUCCUACA | 2419 | UGUAGGRAGCUCAUCUCUCCUAT | 2439

A0-20463.1 | GGAGAGAUGAGCUUCCUACAG | 2420 | CUGUAGGAAGCUCAUCUCUCCUA | 2440

AD-20464.1 | GAGAGAUGAGCUUCCUACAGC | 2421 | GCUGUAGGAAGCUCAUCUCICCY

AD-20465.1 | AGAGAUGAGCUUCCUACAGCA | 2422 | UCCUGUAGGAAGCUCAUCUCUCC | 2442

AD-20466.1 | GAGAUGAGCUUCCUARCAGCAC | 2423 | GUGCUGUAGEGAAGCUCAUCUCUC | 2443

Example 14. Inhibition of EaS/KSP and VEGF expression in humans

A human subject is treated with a pharmaceutical composition, e.g., ALNVSP02, having both a SNALP formulated dsRNA targeted to 2 Eg5/KSP gene and a SNALP formulated dsRNA targeted to a VEGF gene to inhibit expression of the Eg5/KSP and VEGF genes.

A subject in need of treatment is selected or identified. The subject can be in need of cancer treatment, e.g., liver cancer.

At time zero, a suitable first dose of the composition is subcutaneously administered to the subject. The composition is formulated as described herein. After a period of time, the subject’s condition is evaluated, e.g., by measurement of tumor growth, measuring serum

AFP levels, and the like. This measurement can be accompanied by a measurement of

Eg5/KSP and/or VEGF expression in said subject, and/or the products of the successful siRNA-targeting of Eg5/KSP and/or VEGF mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject’s needs.

After treatment, the subject’s condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly affficted but untreated subject.

Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will aliow them to practice this invention to the full scope of the claims hereinafter appended.

Claims (1)

1. CLAIMS We claim:

   1. A composition comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of 2 human kinesin family member 11 {Eg5/KSP) gene ina cell and a second dsRNA for inhibiting expression of a human VEGF in a cell, wherein: both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALPY); said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand comprises a first sequence and said first antisense strand comprises a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1311 (57 ~ UCGAGAAUCUAAACUAACU-3'), wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and said second dsRNA consists of a second sense strand and a second antisense strand, said second sense strand comprising a third sequence and said second antisense strand comprising a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1538 (5"-GCACAUAGGAGAGAUGAGCUU-3' ), wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in Jength.

   2. The composition of claim 1, wherein the first antisense strand comprises a second sequence complementary to SEQ ID NO:1311 (5 ~UCGAGAAUCUAARACUAACU-3') and the second antisense strand comprises a fourth sequence complementary to SEQ ID NO:1538 (5’-GCACAUAGGAGAGAUGAGCUU-3") ..

   3. The composition of claim 1, wherein the first dsRNA consists of a sense strand consisting of SEQ ID NO:1534 (5°-UCGAGAAUCUAARACUAACUTT~3") and an antisense strand consisting of SEQ ID NO:1535 (5’-AGUUAGUUUAGAUUCUCGATT~3") and the second dsRNA consists of a sense strand consisting of SEQ ID NO:1536 (5°- GCACAUAGGAGAGAUGAGCUU-3"}, and an antisense strand consisting of SEQ ID NO:1537 {5’-AAGCUCAUCUCUCCUAUGUGCUG-3" 1}.

   4. The composition of claim 3, wherein each strand is modified as follows to include a 2°-0O- methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s™ the first dsRNA consists of a sense strand consisting of SEQ ID NO:1240 (57 ~-ucGAGAAucuAAAcuAAcuTsT-37)

   and an antisense strand consisting of SEQ ID NO:1241 (5’-AGUuAGUUuAGAUUCUCGATST); the second dsRNA consists of a sense strand consisting of SEQ ID NO:1242 (57-GecAcAuAGGAGAGARGAGCUsU-3") and an antisense strand consisting of SEQ ID NO:1243 (5’-AAGCUcAUCUCUCCuAuGuGCusG-37).

   A. The composition of claims 1, 2, or 3, wherein said first and second dsRNA comprises at least one modified nucleotide.

   6. The composition of claim 5, wherein said modified nucleotide is chosen from the group of: a 2'-O-methy! modified nucleotide, a nucleotide comprising a 5-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

   7. The composition of claim 5, wherein said modified nucleotide is chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino-modified nucleotide, 2’ -alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

   8. The composition of claims |, 2, and 3, wherein said first and second dsRNA each comprise at least one 2'-O-methyl modified ribonucleotide and at least one nucleotide comprising a 5'-phosphorothioate group.

   9. The composition of claims 1-3 and 3-8, wherein each strand of each dsRNA is 19-23 bases in length.

   16. The composition of claims 1-3 and 5-8, wherein each strand of each dsRNA is 21-23 bases in length.

   11. The composition of claims 1-3 and 5-8, wherein each strand of the first dsRNA is 21 bases in length and the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length.

   12. The composition of claims 1-11, wherein the first and second dsRNA are present in an equimolar ratio.

   13. The composition of claims 1-12, wherein said SNALP comprises DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA.

   14. The composition of claims 1-13, comprising the components in the proportions listed in Table 17. 15, The composition of claims 1-14, wherein said composition, upon contact with a cell expressing Eg5, inhibits expression of EgS by at least 40, 50, 60, 70, 80, or by at least 90%.

   16. The composition of claims 1-15, wherein said composition, upon contact with a cell expressing VEGF, inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or by at least

   80%.

   17. The composition of claims 1-16, wherein administration of said composition to a cell decreases expression of both Eg5 and VEGF in said cell.

   18. The composition of claims 1-17, wherein the composition is administered in a 1M concentration.

   19. The composition of claims 1-18, wherein administration of said composition to a cell increases mono-aster formation in the cell.

   20. The composition of claims 1-19, wherein administration of said composition to a mammal results in at least one effect selected from the group consisting of prevention of tumor growth, reduction in tumor growth, or prolonged survival in said mammal.

   21. The composition of claims 1-20, wherein said effect is measured using at least one assay selected from the group consisting of determination of body weight, determination of organ weight, visual inspection, mRNA analysis, serum AFP analysis and survival monitoring.

   22. The composition of claims 1-21, further comprising Sorafenib.

   23. The composition of any of the above claims, wherein the first dsRNA contains two overhangs and the second dsRNA contains an overhang at the 3° of the antisense and a blunt end at the 5° end of the antisense strand.

   24. A method for inhibiting the expression of Eg5/KSP and VEGF in a cell comprising administering any of the compositions of claims 1-22 to the cell.

   25. A method for preventing tumor growth, reducing tumor growth, or prolonging survival in a mammal in need of treatment for cancer comprising administering the composition of claims 1-22 to said mammal.

   26. The method of claim 25, wherein said mammal has liver cancer.

   27. The method of claim 25, wherein said mammal is a human with liver cancer.

   28. The method of claim 24 or 25, further comprising administering Sorafenib.